Inhibitors of serine proteases, particularly hepatitis c virus ns3 protease

ABSTRACT

The present invention relates to compounds, methods and pharmaceutical compositions for inhibiting proteases, particularly serine proteases, and more particularly HCV NS3 proteases. The compounds, and the compositions and methods that utilize them, can be used, either alone or in combination to inhibit viruses, particularly HCV virus.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.10/607,716, filed Jun. 27, 2003, which is a divisional of U.S. patentapplication Ser. No. 09/875,390, filed Jun. 6, 2001, now U.S. Pat. No.6,617,309, which is a divisional of U.S. patent application Ser. No.09/293,247, filed Apr. 16, 1999, now U.S. Pat. No. 6,265,380, which is acontinuation of PCT International Application No. PCT/US97/18968, filedOct. 17, 1997, which claims priority to U.S. Patent Application No.60/028,290, filed Oct. 18, 1996, all of which disclosures areincorporated herein by reference in their entireties.

TECHNICAL FIELD OF THE INVENTION

The present invention relates to a novel class of compounds that areuseful as protease inhibitors, particularly as serine proteaseinhibitors, and more particularly as hepatitis C NS3 proteaseinhibitors. As such, they act by interfering with the life cycle of thehepatitis C virus and are also useful as antiviral agents.

This invention also relates to pharmaceutical compositions comprisingthese compounds. The compounds and pharmaceutical compositions of thisinvention are particularly well suited for inhibiting HCV NS3 proteaseactivity and consequently, may be advantageously used as therapeuticagents against the hepatitis C virus and other viruses that aredependent upon a serine protease for proliferation. This invention alsorelates to methods for inhibiting the activity of proteases, includinghepatitis C virus NS3 protease and other serine proteases, using thecompounds of this invention and related compounds.

BACKGROUND OF THE INVENTION

Infection by hepatitis C virus (“HCV”) is a compelling human medicalproblem. HCV is recognized as the causative agent for most cases ofnon-A, non-B hepatitis, with an estimated human seroprevalence of 1%globally [Purcell, R. H., “Hepatitis C virus: Historical perspective andcurrent concepts” FEMS Microbiology Reviews 14, pp. 181-192 (1994); Vander Poel, C. L., “Hepatitis C Virus. Epidemiology, Transmission andPrevention in Hepatitis C Virus. Current Studies in Hematology and BloodTransfusion, H. W. Reesink, Ed., (Basel: Karger), pp. 137-163 (1994)].Four million individuals may be infected in the United States alone[Alter, M. J. and Mast, E. E., “The Epidemiology of Viral Hepatitis inthe United States, Gastroenterol. Clin. North Am. 23, pp. 437-455(1994)].

Upon first exposure to HCV only about 20% of infected individualsdevelop acute clinical hepatitis while others appear to resolve theinfection spontaneously. In most instances, however, the virusestablishes a chronic infection that persists for decades [Iwarson, S.“The Natural Course of Chronic Hepatitis” FEMS Microbiology Reviews 14,pp. 201-204 (1994)]. This usually results in recurrent and progressivelyworsening liver inflammation, which often leads to more severe diseasestates such as cirrhosis and hepatocellular carcinoma [Kew, M. C.,“Hepatitis C and Hepatocellular Carcinoma”, FEMS Microbiology Reviews,14, pp. 211-220 (1994); Saito, I., et al. “Hepatitis C Virus Infectionis Associated with the Development of Hepatocellular Carcinoma” Proc.Natl. Acad. Sci. USA 87, pp. 6547-6549 (1990)]. Unfortunately, there areno broadly effective treatments for the debilitating progression ofchronic HCV.

The HCV genome encodes a polyprotein of 3010-3033 amino acids [Choo,Q.-L., et al. “Genetic Organization and Diversity of the Hepatitis CVirus”, Proc. Natl. Acad. Sci. USA, 88, pp. 2451-2455 (1991); Kato, N.et al., Molecular Cloning of the Human Hepatitis C Virus Genome FromJapanese Patients with Non-A, Non-B Hepatitis”, Proc. Natl. Acad. Sci.USA, 87, pp. 9524-9528 (1990); Takamizawa, A. et al., “Structure andOrganization of the Hepatitis C Virus Genome Isolated From HumanCarriers”, J. Virol., 65, pp. 1105-1113 (1991)]. The HCV nonstructural(NS) proteins are presumed to provide the essential catalytic machineryfor viral replication. The NS proteins are derived by proteolyticcleavage of the polyprotein [Bartenschlager, R. et al., “NonstructuralProtein 3 of the Hepatitis C Virus Encodes a Serine-Type ProteinaseRequired for Cleavage at the NS3/4 and NS4/5 Junctions”, J. Virol., 67,pp. 3835-3844 (1993); Grakoui, A. et al. “Characterization of theHepatitis C Virus-Encoded Serine Proteinase: Determination ofProteinase-Dependent Polyprotein Cleavage Sites”, J. Virol., 67, pp.2832-2843 (1993); Grakoui, A. et al., Expression and Identification ofHepatitis C Virus Polyprotein Cleavage Products”, J. Virol., 67, pp.1385-1395 (1993); Tomei, L. et al., “NS3 is a serine protease requiredfor processing of hepatitis C virus polyprotein”, J. Virol., 67, pp.4017-4026 (1993)].

The HCV NS protein 3 (NS3) contains a serine protease activity thathelps process the majority of the viral enzymes, and is thus consideredessential for viral replication and infectivity. It is known thatmutations in the yellow fever virus NS3 protease decreases viralinfectivity [Chambers, T. J. et. al., “Evidence that the N-terminalDomain of Nonstructural Protein NS3 From Yellow Fever Virus is a SerineProtease Responsible for Site-Specific Cleavages in the ViralPolyprotein”, Proc. Natl. Acad. Sci. USA, 87, pp. 8898-8902 (1990)]. Thefirst 181 amino acids of NS3 (residues 1027-1207 of the viralpolyprotein) have been shown to contain the serine protease domain ofNS3 that processes all four downstream sites of the HCV polyprotein [C.Lin et al., “Hepatitis C Virus NS3 Serine Proteinase: Trans-CleavageRequirements and Processing Kinetics”, J. Virol., 68, pp. 8147-8157(1994)].

The HCV NS3 serine protease and its associated cofactor, NS4A, helpprocess all of the viral enzymes, and are thus considered essential forviral replication. This processing appears to be analogous to thatcarried out by the human immunodeficiency virus aspartyl protease, whichis also involved in viral enzyme processing. HIV protease inhibitors,which inhibit viral protein processing, are potent antiviral agents inman, indicating that interrupting this stage of the viral life cycleresults in therapeutically active agents. Consequently it is anattractive target for drug discovery. Unfortunately, there are no serineprotease inhibitors available currently as anti-HCV agents.

Furthermore, the current understanding of HCV has not led to any othersatisfactory anti-HCV agents or treatments. The only established therapyfor HCV disease is interferon treatment. However, interferons havesignificant side effects (Janssen et al., 1994; Renault and Hoofnagle,1989) [Janssen, H. L. A., et al. “Suicide Associated withAlfa-Interferon Therapy for Chronic Viral Hepatitis” J. Hepatol., 21,pp. 241-243 (1994)]; Renault, P. F. and Hoofnagle, J. H., “Side effectsof alpha interferon. Seminars in Liver Disease 9, 273-277. (1989)] andinduce long term remission in only a fraction (˜25%) of cases [Weiland,O. “Interferon Therapy in Chronic Hepatitis C Virus Infection”, FEMSMicrobiol. Rev., 14, PP. 279-288 (1994)]. Moreover, the prospects foreffective anti-HCV vaccines remain uncertain.

Thus, there is a need for more effective anti-HCV therapies. Suchinhibitors would have therapeutic potential as protease inhibitors,particularly as serine protease inhibitors, and more particularly as HCVNS3 protease inhibitors. Specifically, such compounds may be useful asantiviral agents, particularly as anti-HCV agents.

SUMMARY OF THE INVENTION

The present invention provides compounds, and pharmaceuticallyacceptable derivatives thereof, that are useful as protease inhibitors,particularly as serine protease inhibitors, and more particularly as HCVNS3 protease inhibitors. These compounds can be used alone or incombination with immunomodulatory agents, such as α-, β- orγ-interferons; other antiviral agents such as ribavirin and amantadine;other inhibitors of hepatitis C protease; inhibitors of other targets inthe HCV life cycle including the helicase, the polymerase, themetalloprotease, or the internal ribosome entry; or combinationsthereof.

The present invention also provides methods for inhibiting proteases,particularly serine proteases, and more particularly HCV NS3 protease.

The present invention also provides pharmaceutical compositionscomprising the compounds of this invention, as well as multi-componentcompositions comprising additional immunomodulatory agents, such as α-,β- or γ-interferons; other antiviral agents such as ribavirin andamantadine; other inhibitors of hepatitis C protease; inhibitors ofother targets in the HCV life cycle including the helicase, thepolymerase, the metalloprotease or the internal ribosome entry; orcombinations thereof. The invention also provides methods of using thecompounds of this invention, as well as other related compounds, for theinhibition of HCV.

DETAILED DESCRIPTION OF THE INVENTION

In order that the invention herein described may be more fullyunderstood, the following detailed description is set forth. In thedescription, the following abbreviations are used:

Designation Reagent or Fragment Abu aminobutyric acid Ac acetyl AcOHacetic acid Bn benzyl Boc tert-butyloxycarbonyl Bz benzoyl Cbzcarbobenzyloxy CDI carbonyldiimidazole DCE 1,2-dichloroethane DCMdichlorome thane DIEA diisopropylethylamine DMA dimethylacetamide DMAPdimethylaminopyridine DMF dimethylformamide DPPA diphenylphosphorylazideDMSO dimethylsulfoxide Et ethyl EtOAc ethyl acetate FMOC9-fluorenylmethoxycarbonyl HbtU O-benzotriazolyl-N,N,N′,N′-tetramethyluronium hexafluorophosphate HOBt N-hydroxybenzotriazole HPLChigh performance liquid chromatography Me methyl MS mass spectrometryNMP N-methyl pyrrolidinone ND not determined Pip piperidine Przpiperazine PyBrop bromo-tris-pyrrolidinophosphonium hexafluorophosphatePyr pyridine THF tetrahydrofuran TFA trifluoroacetic acid TFEtrifluoroethanol Tol toluene

The following terms are used herein:

Unless expressly stated to the contrary, the terms “—SO₂-” and “—S(O)₂-”as used herein refer to a sulfone or sulfone derivative (i.e., bothappended groups linked to the S), and not a sulfinate ester.

The term “substituted” refers to the replacement of one or more hydrogenradicals in a given structure with a radical selected from a specifiedgroup. When more than one hydrogen radical may be replaced with asubstituent selected from the same specified group, the substituents maybe either the same or different at every position.

As used herein, the term “amino” refers to a trivalent nitrogen whichmay be primary or which may be substituted with 1-2 alkyl groups.

The term “alkyl” or “alkane”, alone or in combination with any otherterm, refers to a straight-chain or branched-chain saturated aliphatichydrocarbon radical containing the specified number of carbon atoms, orwhere no number is specified, preferably from 1-10 and more preferablyfrom 1-5 carbon atoms. Examples of alkyl radicals include, but are notlimited to, methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl,sec-butyl, tert-butyl, pentyl, isoamyl, n-hexyl and the like.

The term “alkenyl” or “alkene”, alone or in combination with any otherterm, refers to a straight-chain or branched-chain mono- orpoly-unsaturated aliphatic hydrocarbon radical containing the specifiednumber of carbon atoms, or where no number is specified, preferably from2-10 carbon atoms and more preferably, from 2-5 carbon atoms. Examplesof alkenyl radicals include, but are not limited to, ethenyl, E- andZ-propenyl, E- and Z-isobutenyl, E- and Z-pentenyl, E- and Z-hexenyl,E,E-, E,Z-, Z,E-, and Z-Z-hexadienyl and the like.

The term “alkynyl” or “alkyne”, alone or in combination with any otherterm, refers to a straight-chain or branched-chain mono orpoly-unsaturated aliphatic hydrocarbon radical containing the specifiednumber of carbon atoms, or where no number is specified, preferably from2-10 carbon atoms and more preferably, from 2-5 carbon atoms, wherein atleast one of the unsaturated aliphatic hydrocarbon radicals comprises atriple bond. Examples of alkynyl radicals include, but are not limitedto, ethynyl, propynyl, isobutynyl, pentynyl, hexynyl, hexenynyl, and thelike.

The term “aryl”, alone or in combination with any other term, refers toa carbocyclic aromatic radical containing the specified number of carbonatoms, and which may be optionally fused, for example benzofused, withone to three cycloalkyl, aromatic, heterocyclic or heteroaromatic rings.Preferred aryl groups have from 6-14 carbon atoms, and more preferredgroups from 6-10 carbon atoms. Examples of aryl radicals include, butare not limited to, phenyl, naphthyl, anthracenyl and the like.

The term “carbocycle”, alone or in combination with any other term,refers to a stable non-aromatic 3- to 8-membered carbon ring radicalwhich may be saturated, mono-unsaturated or poly-unsaturated, and whichmay be optionally fused, for example benzofused, with one to threecycloalkyl, aromatic, heterocyclic or heteroaromatic rings. Thecarbocycle may be attached at any endocyclic carbon atom which resultsin a stable structure.

The terms “cycloalkyl” or “cycloalkane”, alone or in combination withany other term, refers to a stable non-aromatic 3- to 8-membered carbonring radical which is saturated and which may be optionally fused, forexample benzofused, with one to three cycloalkyl, aromatic, heterocyclicor heteroaromatic rings. The cycloalkyl may be attached at anyendocyclic carbon atom which results in a stable structure. Preferredcarbocycles have 5 to 6 carbons. Examples of carbocycle radicalsinclude, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl,cyclohexyl, cycloheptyl, cyclopentenyl, cyclohexenyl, indane,tetrahydronaphthalene and the like.

The term “cycloalkenyl” or “cycloalkene” alone or in combination withany other term, refers to a stable cyclic hydrocarbon ring radicalcontaining at least one endocyclic carbon-carbon double bond. Thecarbocycle may be attached at any cyclic carbon atom which results in astable structure. Where no number of carbon atoms is specified, acycloalkenyl radical preferably has from 5-7 carbon atoms. Examples ofcycloalkenyl radicals include, but are not limited to, cyclopentenyl,cyclohexenyl, cyclopentadienyl, indenyl and the like.

The term “cycloalkylidenyl”, alone or in combination with any otherterm, refers to a stable cyclic hydrocarbon ring radical containing atleast one exocyclic carbon-carbon double bond, wherein the cyclichydrocarbon ring may be optionally fused, for example benzofused, withone to three cycloalkyl, aromatic, heterocyclic or heteroaromatic rings.The carbocycle may be attached at any cyclic carbon atom, which resultsin a stable structure. Where no number of carbon atoms is specified, acycloalkylidenyl radical preferably has from 5-7 carbon atoms. Examplesof cycloalkylidenyl radicals include, but are not limited to,cyclopentylidenyl, cyclohexylidenyl, cyclopentenylidenyl and the like.

The skilled practitioner would realize that certain groups could beclassified either as cycloalkanes or as aryl groups. Examples of suchgroups include indanyl and tetrahydro naphthyl groups.

The term “monocycle” or “monocyclic” alone or in combination with anyother term, unless otherwise defined herein, refers to a 5-7 memberedring system.

The term “bicycle” or “bicyclic” alone or in combination with any otherterm, unless otherwise defined herein, refers to a 6-11 membered ringsystem.

The term “tricycle” or “tricyclic” alone or in combination with anyother term, unless otherwise defined herein, refers to a 11-15 memberedring system.

The terms “heterocyclyl” and “heterocycle”, alone or in combination withany other term, unless otherwise defined herein, refers to a stable 5-to 15-membered mono-, bi-, or tricyclic, heterocyclic ring which iseither saturated or partially unsaturated, but not aromatic, and whichmay be optionally fused, for example benzofused, with one to threecycloalkyl, aromatic, heterocyclic or heteroaromatic rings. Eachheterocycle consists of one or more carbon atoms and from one to fourheteroatoms selected from the group consisting of nitrogen, oxygen andsulfur. As used herein, the terms “nitrogen and sulfur heteroatoms”include any oxidized form of nitrogen and sulfur, and the quaternizedform of any basic nitrogen. A heterocycle may be attached at anyendocyclic carbon or heteroatom which results in the creation of astable structure.

Preferred heterocycles defined above include, for example,imidazolidinyl, indazolinolyl, perhydropyridazyl, pyrrolinyl,pyrrolidinyl, piperidinyl, pyrazolinyl, piperazinyl, morpholinyl,thiamorpholinyl, β-carbolinyl, thiazolidinyl, thiamorpholinyl sulfone,oxopiperidinyl, oxopyrrolidinyl, oxoazepinyl, azepinyl, furazanyl,tetrahydropyranyl, tetrahydrofuranyl, oxathiolyl, dithiolyl,tetrahydrothiophenyl, dioxanyl, dioxolanyl,tetrahydrofurotetrahydrofuranyl, tetrahydropyranotetrahydrofuranyl,tetrahydrofurodihydrofuranyl, tetrahydropyranodihydrofuranyl,dihydropyranyl, dihydrofuranyl, dihydrofurotetrahydrofuranyl,dihydropyranotetrahydrofuranyl, sulfolanyl and the like.

The terms “heteroaryl” and “heteroaromatic” alone or in combination withany other term, unless otherwise defined herein, refers to a stable 3-to 7-membered monocyclic heterocyclic ring which is aromatic, and whichmay be optionally fused, for example, benzofused, with one to threecycloalkyl, aromatic, heterocyclic or heteroaromatic rings. Eachheteroaromatic ring consists of one or more carbon atoms and from one tofour heteroatoms selected from the group consisting of nitrogen, oxygenand sulfur. As used herein, the terms “nitrogen and sulfur heteroatoms”include any oxidized form of nitrogen and sulfur, and the quaternizedform of any basic nitrogen. A heteroaromatic ring may be attached at anyendocyclic carbon or heteroatom which results in the creation of astable, aromatic structure.

Preferred heteroaromatics defined above include, for example,benzimidazolyl, imidazolyl, quinolyl, isoquinolyl, indolyl, indazolyl,pyridazyl, pyridyl, pyrrolyl, pyrazolyl, pyrazinyl, quinoxolyl, pyranyl,pyrimidinyl, pyridazinyl, furyl, thienyl, triazolyl, thiazolyl,tetrazolyl, benzofuranyl, oxazolyl, benzoxazolyl, isoxazolyl,isothiazolyl, thiadiazolyl, thiophenyl, and the like.

The term “halo” refers to a radical of fluorine, chlorine, bromine oriodine. Preferred halogen radicals include fluorine and chlorine.

In chemical formulas, parentheses are used herein to indicate 1) thepresence of more than one atom or group bonded to the same atom orgroup; or 2) a branching point in a chain (i.e., the group or atomimmediately before the open parenthesis is bonded directly to the groupor atom immediately after the closed parenthesis). An example of thefirst use is “N(R¹)₂” denoting two R¹ groups bound to the nitrogen atom.An example of the second use is “—C(O)R¹” denoting an oxygen atom and aR¹ bound to the carbon atom, as in the following structure:

As used herein, “B” indicates a boron atom.

The present invention provides compounds that are useful as proteaseinhibitors, particularly as serine protease inhibitors, and moreparticularly as HCV NS3 protease inhibitors. As such, they act byinterfering with the life cycle of the HCV virus and other viruses thatare dependent upon a serine protease for proliferation. Therefore, thesecompounds are useful as antiviral agents.

Accordingly, in one embodiment, the present invention provides acompound of the formula (I):

wherein:

G¹ is thiol, hydroxyl, thiomethyl, alkenyl, alkynyl, trifluoromethyl,C₁₋₂ alkoxy, C₁₋₂ alkylthio, or C₁₋₃ alkyl, wherein the C₁₋₃ alkyl groupis optionally substituted with thiol, hydroxyl, thiomethyl, alkenyl,alkynyl, trifluoromethyl, C₁₋₂ alkoxy, or C₁₋₂ alkylthio.

W¹ is:

G² is alkyl, aryl, aralkyl, or a mono-, bi- or tricyclic heterocycle,optionally substituted with 1-3 groups selected from alkyl, alkenyl,alkynyl, aralkyl, alkoxy, alkenoxy, aryloxy, heterocyclyl,heterocyclylalkyl, aralkoxy, heterocyclylalkoxy, oxo, hydroxy, amino,alkanoylamino, alkoxycarbonylamino, ureido, carboxy,heterocyclyloxyalkyl, aryloxyalkyl, heterocyclylcarbonyl, aroyl,arylsulfonyl, heterocyclylsulfonyl, heterocyclylsulfonylamino,arylsulfonamido, aralkylsulfonamido, heterocyclylalkanoyl, carboxyalkyl,carboxyamidoalkyl, alkanesulfonyl, sulfonamido, halo, cyano, orhaloalkyl.

G⁴ is alkyl, alkenyl, alkynyl, cycloalkyl, cycloalkylalkyl,cycloalkylalkenyl, aryl, aralkyl, aralkenyl, heterocyclyl,heterocyclylalkyl, heterocyclylalkenyl, hydroxyalkyl, alkoxyalkyl,alkylthioalkyl, arylthioalkyl, or heterocyclylthioalkyl.

Each Q¹ is hydroxy, alkoxy, or aryloxy, or each Q¹ is an oxygen atom andtogether with the boron to which they are bound, form a 5-7 memberedring, wherein the ring atoms are carbon, nitrogen or oxygen.

U is hydrogen, G⁹-C(O)—, G⁹-SO₂—, G⁹-C(O)—C(O)—, (G⁹)₂-N—C(O)—C(O)—,(G⁹)₂-N—SO₂—, (G⁹)₂N—C(O)—, or G⁹-O—C(O)—.

G⁹ is hydrogen, alkyl, carboxyalkyl, alkenyl, aryl, aralkyl, aralkenyl,cycloalkyl, cycloalkylalkyl, cycloalkenyl, heterocyclyl,heterocyclylalkyl, or heterocyclyalkenyl optionally substituted with 1-3groups selected from alkyl, alkenyl, aralkyl, alkoxy, alkenoxy, aryloxy,heterocyclyl, carboxyalkyl, carboxyamidoalkyl, alkylsulfonyl, orsulfonamido; or two G⁹ groups, together with the nitrogen atom to whichthey are bound, form a 4-10 membered nitrogen containing monocyclic orbicyclic saturated or partially unsaturated ring system, wherein 1-2 ofthe atoms forming the ring are N, S, or O and the other atoms formingthe ring are C; wherein the ring system is optionally substituted by oneor two groups selected from alkyl, alkenyl, aralkyl, alkoxy, alkenoxy,aryloxy, aralkoxy, heterocyclyl, keto, hydroxy, amino, alkanoyl amino,carboxy, carboxyalkyl, carboxamidoalkyl, sulfonyl, or sulfonamido.

E⁴ is a bond;

wherein:

G¹³ is cycloalkylalkyl, aralkyl, heterocycylalkyl, aralkoxyalkyl,heterocycylalkoxyalkyl, aralkylthioalkyl, or heterocycylalkylthioalkyl,optionally substituted by 1-2 alkyl, alkenyl, aralkyl, alkoxy, alkenoxy,aryloxy, aralkoxy, heterocyclyl, oxo, hydroxy, amino, alkanoylamino,carboxy, carboxyalkyl, carboxamidoalkyl, sulfonyl, or sulfonamidogroups.

G¹⁴ is hydrogen, alkyl, alkenyl, hydroxy, alkoxy, or —CH₂-G⁸, wherein G⁸is aryl, aralkyl, carbocyclyl or heterocyclyl, where the ring portion ofeach aryl, aralkyl, or heterocycle is optionally substituted with 1-3groups selected from alkyl, alkenyl, aralkyl, alkoxy, alkenoxy, aryloxy,heterocyclyl, heterocyclylalkyl, aralkoxy, heterocyclylalkoxy, oxo,hydroxy, amino, alkanoylamino, alkoxycarbonylamino, ureido, carboxy,carboxyalkyl, carboxyamidoalkyl, alkanesulfonyl, sulfonamido, halo,cyano, or haloalkyl; or

when E⁴ is:

G¹³ and G¹⁴, together with the atoms to which they are bound (carbon andnitrogen, respectively), form a nitrogen-containing heterocyclic ringsystem having 4-7 members, which optionally contains one to twoadditional heteroatoms, wherein the resulting ring system is optionallyfused with an additional carbocyclic or heterocyclic ring system to forma bicyclic ring system comprising 7-11 atoms; and wherein the monocyclicor bicyclic ring system is optionally substituted by one or two groupsselected from oxo, hydroxy, alkyl, alkenyl, aryl, aralkyl, alkyl,alkenoxy, aryloxy, aralkyloxy, halo, or nitro.

Each Q³ is halo, nitro, cyano, alkyl, alkenyl, aralkyl, alkoxy,alkenoxy, aryloxy, aralkoxy, heterocyclyl, heterocyclylalkyl, hydroxy,amino, alkylamino, alkanoylamino, carboxy, carboxyalkyl,carboxamidoalkyl, alkylsulfonyl, arylsulfonyl, aralkylsulfonyl,alkylsulfonamido, arylsulfonamido, or aralkylsulfonamido, wherein anyalkyl, alkenyl, aryl, or heterocyclyl groups is optionally substitutedwith 1-3 groups selected from keto, hydroxy, nitro, cyano, halo, amino,alkyl, alkoxy, or alkylthio; wherein Q³, when not bonded to a specificatom, may be bonded to any substitutable atom.

Q⁴ is independently alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, alkanoyl, arylcarbonyl, aralkylcarbonyl,alkoxycarbonyl, aryloxycarbonyl, aralkyloxycarbonyl, alkylsulfonyl,arylsulfonyl, aralkylsulfonyl, alkylaminocarbonyl, arylaminocarbonyl,aralkylaminocarbonyl, wherein any of said alkyl, cycloalkyl, aryl,aralkyl, heterocyclyl groups is optionally substituted with one or moregroups independently selected from keto, hydroxy, nitro, cyano, halo,amino, alkyl, alkoxy, or alkylthio.

Q⁵ is aryl or an aromatic heterocycle, wherein:

the aryl or aromatic heterocycle is monocyclic, bicyclic, or tricyclichaving 5-14 atoms, and is optionally substituted with 1-3 groupsselected from hydroxy, nitro, cyano, halo, amino, alkyl, alkoxy,alkanoyl, alkylamino, or alkylthio.

E⁵ is a bond or

wherein G¹⁵ is alkyl, alkenyl, cycloalkylalkyl, aralkyl,heterocyclylalkyl, carboxyalkyl, or carboxamidoalkyl, where the ring ofany aralkyl or heterocyclylalkyl group is optionally substituted with1-2 alkyl, alkenyl, aralkyl, alkoxy, alkenoxy, aryloxy, aralkoxy,heterocyclyl, oxo, hydroxy, amino, alkanoylamino, carboxy, carboxyalkyl,carboxamidoalkyl, sulfonyl, or sulfonamido groups.

E⁶ is a bond or

wherein G¹⁶ is hydrogen, alkyl, alkenyl, aralkyl, or cycloalkylalkylyl;

E⁷ is a bond or

wherein G¹⁷ is alkyl optionally substituted with carboxy; wherein thealkyl is preferably C₁₋₃ alkyl.

E⁸ is a bond or

wherein G¹⁸ is alkyl optionally substituted with carboxy; wherein thealkyl is preferably C₁₋₃ alkyl.

Each Z¹ is independently O or H₂ provided that no more than two Z¹groups is H₂ in a given compound.

Preferred compounds of formula (I) are those in which at least onesubstituent is defined as follows:

G¹ is vinyl, acetylenyl, —CH₃, —CF₃, —CH₂CH₃, —CH₂CF₃, —SCH₃, —SH,—CH₂SH, or —CH₂OH;

G¹³ is C₃₋₆ branched alkyl or G¹³ and G¹⁴, together with the atoms towhich they are bound (carbon and nitrogen, respectively), form anitrogen-containing heterocyclic ring system having 4-7 members, whichoptionally contains one to two additional heteroatoms, wherein themonocyclic or bicyclic ring system is optionally substituted by one ortwo groups selected from oxo, hydroxy, alkyl, alkenyl, aryl, aralkyl,alkyl, alkenoxy, aryloxy, aralkyloxy, halo, or nitro; and

Z¹ is O.

More preferred compounds of formula (I) are those wherein G¹ is —SH,—CH₂SH, —CF₃, or —CF₂CF₃.

Most preferred compounds of formula (I) are those wherein G¹ is —SH or—CF₃.

According to another embodiment, the present invention provides acompound of the formula (I), wherein W¹ is as defined below for W andthe other substituents are as defined above.

According to another embodiment, the present invention provides acompound of the formula (II):

In these compounds:

W is:

m is 0 or 1.

Each R¹ is hydroxy, alkoxy, or aryloxy, or each R¹ is an oxygen atom andtogether with the boron, to which they are each bound, form a 5-7membered ring, wherein the ring atoms are carbon, nitrogen, or oxygen.

Each R² is independently hydrogen, alkyl, alkenyl, aryl, aralkyl,aralkenyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, cycloalkenylalkyl,heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heteroaryl, orheteroaralkyl, or two R² groups, which are bound to the same nitrogenatom, form together with that nitrogen atom, a 5-7 membered monocyclicheterocyclic ring system; wherein any R² carbon atom is optionallysubstituted with J.

J is alkyl, aryl, aralkyl, alkoxy, aryloxy, aralkoxy, cycloalkyl,cycloalkoxy, heterocyclyl, heterocyclyloxy, heterocyclylalkyl, keto,hydroxy, amino, alkylamino, alkanoylamino, aroylamino, aralkanoylamino,carboxy, carboxyalkyl, carboxamidoalkyl, halo, cyano, nitro, formyl,acyl, sulfonyl, or sulfonamido and is optionally substituted with 1-3 J¹groups.

J¹ is alkyl, aryl, aralkyl, alkoxy, aryloxy, heterocyclyl,heterocyclyloxy, keto, hydroxy, amino, alkanoylamino, aroylamino,carboxy, carboxyalkyl, carboxamidoalkyl, halo, cyano, nitro, formyl,sulfonyl, or sulfonamido.

L is alkyl, alkenyl, or alkynyl, wherein any hydrogen bound to a carbonatoms is optionally substituted with halogen, and wherein any hydrogenor halogen atom bound to any terminal carbon atom is optionallysubstituted with sulfhydryl or hydroxy.

A¹ is a bond,

R⁴ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroaralkyl, carboxyalkyl, or carboxamidoalkyl, and isoptionally substituted with 1-3 J groups.

R⁵ and R⁶ are independently hydrogen, alkyl, alkenyl, aryl, aralkyl,aralkenyl, cycloalkyl, cycloalkylalkyl, cycloalkenyl, heterocyclyl,heterocyclylalkyl, heteroaryl, or heteroaralkyl, and is optionallysubstituted with 1-3 J groups.

X is a bond, —C(H)(R⁷)—, —O—, —S—, or —N(R⁸)—.

R⁷ is hydrogen, alkyl, alkenyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, or heteroaralkyl, and is optionallysubstituted with 1-3 J groups.

R⁸ is hydrogen alkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroaralkyl, aralkanoyl, heterocyclanoyl,heteroaralkanoyl, —C(O)R¹⁴, —SO₂R¹⁴, or carboxamido, and is optionallysubstituted with 1-3 J groups; or R⁸ and Z, together with the atoms towhich they are bound, form a nitrogen containing mono- or bicyclic ringsystem optionally substituted with 1-3 J groups.

R¹⁴ is alkyl, aryl, aralkyl, heterocyclyl, heterocyclyalkyl, heteroaryl,or heteroaralkyl.

Y is a bond, —CH₂—, —C(O)—, —C(O)C(O)—, —S(O)—, —S(O)₂—, or —S(O)(NR⁷)—,wherein R⁷ is as defined above.

Z is alkyl, aryl, aralkyl, cycloalkyl, cycloalkylalkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroaralkyl, —OR², or —N(R²)₂, whereinany carbon atom is optionally substituted with J, wherein R² is asdefined above.

A² is a bond or

R⁹ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, heteroaralkyl, carboxyalkyl, or carboxamidoalkyl, and isoptionally substituted with 1-3 J groups.

M is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl, heterocyclylalkyl,heteroaryl, or heteroaralkyl, and is optionally substituted by 1-3 Jgroups, wherein any alkyl carbon atom may be replaced by a heteroatom.

V is a bond, —CH₂—, —C(H)(R¹¹)—, —O—, —S—, or —N(R¹¹)—.

R¹¹ is hydrogen or C₁₋₃ alkyl.

K is a bond, —O—, —S—, —C(O)—, —S(O)—, —S(O)₂—, or —S(O)(NR¹¹)—, whereinR¹¹ is as defined above.

T is —R¹², -alkyl-R¹², -alkenyl-R¹², -alkynyl-R¹², —OR¹², —N(R¹²)₂,—C(O)R¹², —C(═NOalkyl)R¹², or

Each R¹² is hydrogen, aryl, heteroaryl, cycloalkyl, heterocyclyl,cycloalkylidenyl, or heterocycloalkylidenyl, and is optionallysubstituted with 1-3 J groups, or a first R¹² and a second R¹², togetherwith the nitrogen to which they are bound, form a mono- or bicyclic ringsystem optionally substituted by 1-3 J groups.

R¹⁰ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, orcarboxamidoalkyl, and is optionally substituted with 1-3 J groups.

R¹⁵ is alkyl, cycloalkyl, aryl, aralkyl, heterocyclyl,heterocyclylalkyl, heteroaryl, heteroaralkyl, carboxyalkyl, orcarboxamidoalkyl, and is optionally substituted with 1-3 J groups.

R¹⁶ is hydrogen, alkyl, aryl, heteroaryl, cycloalkyl, or heterocyclyl.

Preferably, W is

More preferably, W is

Most preferably, W is

wherein R² is aralkyl; or

Preferably, J is alkyl, alkoxy, aryloxy, aryl, aralkyl, aralkoxy, halo,heteroaryl, cyano, amino, nitro, heterocyclyl, acyl, carboxy,carboxyalkyl, alkylamino, hydroxy, heterocyclylalkyl, aralkanoylamino,aroylamino, alkanoylamino, formyl or keto.

More preferably, J is t-butyl, methyl, trifluoromethyl, methoxy, ethoxy,trifluoromethoxy, carboxy, phenyl, benzyl, phenoxy, benzyloxy, fluoro,chloro, bromo, isoxazolyl, pyridinyl, piperidinyl, carboxymethyl,carboxyethyl; dialkylamino, morpholinylmethyl, phenylacetylamino, oracylamino.

Preferably, J¹ is alkoxy, alkyl, halo or aryl.

More preferably, J¹ is C₁₋₃ alkoxy, chloro, C₁₋₃alkyl, or phenyl.

Preferably, L is alkyl, alkenyl, allyl, or propargyl.

More preferably, L is trihalomethyl, sulfhydryl or alkyl substitutedwith trihalomethyl, sulfhydryl, or hydroxy.

Preferably, R⁴ is alkyl, aralkyl, or cycloalkylalkyl, or cycloalkyl.More preferably, R⁴ is phenylalkyl or cycloalkyl. Most preferably, R⁴ isisobutyl, cyclohexylalkyl, or phenethyl.

Preferably, R⁵ and R⁶ are each hydrogen.

Preferably, X is —O— or —N(R⁸)—.

Preferably, R⁸ is hydrogen.

Preferably, Y is —CH₂—, —C(O)—, —C(O)C(O)—, or —S(O)₂—.

Preferably, R² is H, fluorine, trifluoromethyl, alkyl, aryl, aralkyl,heteroaralkyl, heterocyclyl, or heterocyclylalkyl.

Preferably, Z is alkyl, aryl, aralkyl, heterocyclyl, cycloalkyl,heteroaryl, OR², or N(R²)₂, wherein R² is preferably aralkyl or alkenyl.

More preferably, Z is phenyl, 1,4-benzodioxanyl, 1,3-benzodioxolyl,benzothiazolyl, naphthyl, benzyl, oxadiazolyl, isoxazolyl, quinolyl,benzothiophenyl, thiazolyl, cyclohexyl, butyl, naphthyl, dioxolanyl,benzyl, pyridinyl, morpholinyl, N-anilinyl, N-aminobenzothiazole,N-aminobenzodioxole, N-aminonapthylene, N-benzylamine, N-aminopyridine,benzyloxy, allyloxy, or phenethyl, and is optionally substituted with J.

Most preferably, Z is naphthyl, 3,4-dichlorophenyl,2-carbomethoxyphenyl.

Preferably, R⁹ is alkyl. More preferably, R⁹ is propyl. Most preferably,R⁹ is isopropyl.

Preferably, M is alkyl, heteroaralkyl, aryl, cycloalkylalkyl, aralkyl,or aralkyl, wherein one of the alkyl carbon atoms is replaced by O or S.

More preferably M is propyl, methyl, pyridylmethyl, benzyl,naphthylmethyl, phenyl, imidazolylmethyl, thiophenylmethyl,cyclohexylmethyl, phenethyl, benzylthiomethyl, or benzyloxyethyl.

Preferably, V is —N(R¹¹)—.

Preferably, R¹¹ is hydrogen.

Preferably, K is —C(O)— or —S(O)₂—.

Preferably, T is —R¹², -alkyl-R¹², -alkenyl-R¹², —OR¹², —N(R¹²)₂,—C(═NOalkyl)R¹², or

More preferably, T is —R¹² or -alkyl-R¹².

Preferably, R¹² is aryl or heteroaryl and is optionally substituted by1-3 J groups. More preferably, R¹² is 1-naphthyl, isoquinolyl, indolyl,or 2-alkoxy-1-naphthyl.

Preferably, R¹⁰ is alkyl substituted with carboxy. More preferably, R¹⁰is C₁₋₃ alkyl substituted with carboxy.

Preferably, R¹⁵ is alkyl substituted with carboxy. More preferably, R¹⁵is C₁₋₃ alkyl substituted with carboxy.

In a preferred embodiment of formula (II), A¹ is:

A² is a bond.

Preferably, in this preferred embodiment, X is O.

More preferably, Y is —CH₂—.

Alternatively, Y is —C(O)—.

Alternatively, Y is —C(O)— and Z is —N(R²)₂.

Alternatively, in this preferred embodiment, X is —N(R⁸)—.

More preferably, Y is —C(O)—.

Alternatively, Y is —S(O)₂—.

Alternatively, Y is —C(O)— and Z is —N(R²)₂.

Alternatively, in this preferred embodiment, X is —N(R⁸)—, wherein R⁸ is—C(O)R¹⁴ or —S(O)₂R¹⁴.

More preferably, when R⁸ is —C(O)R¹⁴, Y is —C(O)—.

Alternatively, Y is —S(O)₂—.

Alternatively, Y is —C(O)— and Z is —N(R²)₂.

More preferably, when R⁸ is —S(O)₂R¹⁴, Y is —C(O)— and Z is —N(R²)₂.

In a more preferred embodiment of this invention are compounds offormula (II), wherein A₁ is:

wherein, X is —O— and Y is —CH₂—;

A² is

V is —(NR¹¹)—, and

K is —C(O)—.

In another more preferred embodiment of this invention are compounds offormula (II), wherein A¹ is:

wherein, X is O and Y is CH₂,

A² is:

V is —N(R¹¹)—, and

K is —S(O)₂—.

In another more preferred embodiment of this invention are compounds offormula (II), wherein A¹ is:

wherein, X is O and Y is a —CH₂—, A² is a bond;

V is —N(R¹¹)—, and

K is —C(O)—.

In another more preferred embodiment of this invention are compounds offormula (II), wherein A¹ is:

wherein, X is O and Y is —CH₂—,

A² is a bond;

V is —N(R¹¹)—, and

K is —S(O)₂—

Preferably, in these more preferred embodiments, W is:

More preferably, W is

Most preferably, W is

wherein R² is aralkyl; or

Preferably, in these more preferred embodiments, L is alkyl, alkenyl,allyl, or propargyl.

More preferably, L is trihalomethyl, sulfhydryl or alkyl substitutedwith trihalomethyl, sulfhydryl, or hydroxy.

In another preferred embodiment of formula (II), A¹ is

A² is a bond. Preferred groups in this preferred embodiment are asdescribed above.

In another preferred embodiment of formula (II), A¹ is a bond. Preferredgroups in this preferred embodiment are as described above.

In another preferred embodiment of formula (II), A¹ is

The preferred, more preferred, and most preferred groups of thispreferred embodiment are as described above.

This invention anticipates that many active-site directed inhibitors ofthe NS3 protease may be peptidomimetic in nature and thus may bedesigned from the natural substrate. Therefore, preferred substituentsin peptidomimetic inhibitors of this invention include those whichcorrespond to the backbone or side chains of naturally occurringsubstrates or synthetic substrates with high affinity for the enzyme(low K_(m)).

In another preferred embodiment of formula (II), A¹ is a bond. Thepreferred, more preferred, and most preferred compounds of thispreferred embodiment are as described above.

The skilled practitioner would realize that some certain groups could beclassified either as heterocycles or heteroaromatics, depending on thepoint of attachment.

The compounds of this invention may contain one or more asymmetriccarbon atoms and thus may occur as racemates and racemic mixtures,single enantiomers, diastereomeric mixtures and individualdiastereomers. All such isomeric forms of these compounds are expresslyincluded in the present invention. Each stereogenic carbon may be of theR or S configuration. Combinations of substituents and variablesenvisioned by this invention are only those that result in the formationof stable compounds. The term “stable”, as used herein, refers tocompounds which possess stability sufficient to allow manufacture andwhich maintain their integrity for a sufficient period of time to beuseful for the purposes detailed herein (e.g., therapeutic orprophylactic administration to a mammal or for use in affinitychromatography applications). Typically, such compounds are stable at atemperature of 40° C. or less, in the absence of moisture or otherchemically reactive conditions, for at least a week.

The compounds of this invention may be synthesized using conventionaltechniques. Advantageously, these compounds are conveniently synthesizedfrom readily available starting materials.

As used herein, the compounds of this invention, including the compoundsof formulae (I) and (II), are defined to include pharmaceuticallyacceptable derivatives or prodrugs thereof. A “pharmaceuticallyacceptable derivative or prodrug” means any pharmaceutically acceptablesalt, ester, salt of an ester, or other derivative of a compound of thisinvention which, upon administration to a recipient, is capable ofproviding (directly or indirectly) a compound of this invention.

Accordingly, this invention also provides prodrugs of the compounds ofthis invention, which are derivatives that are designed to enhancebiological properties such as oral absorption, clearance, metabolism orcompartmental distribution. Such derivatives are well known in the art.

As the skilled practitioner realizes, the compounds of this inventionmay be modified by appending appropriate functionalities to enhanceselective biological properties. Such modifications are known in the artand include those which increase biological penetration into a givenbiological compartment (e.g., blood, lymphatic system, central nervoussystem), increase oral availability, increase solubility to allowadministration by injection, alter metabolism and alter rate ofexcretion.

The term “protected” refers to when the designated functional group isattached to a suitable chemical group (protecting group). Examples ofsuitable protecting groups are described in T. W. Greene and P. G. M.Wuts, Protective Groups in Organic Synthesis, 2d. Ed., John Wiley andSons (1991); L. Fieser and M. Fieser, Fieser and Fieser's Reagents forOrganic Synthesis, John Wiley and Sons (1994); L. Paquette, ed.Encyclopedia of Reagents for Organic Synthesis, John Wiley and Sons(1995) and are exemplified in certain of the specific compounds used inthis invention.

Particularly favored derivatives and prodrugs are those that increasethe bioavailability of the compounds of this invention when suchcompounds are administered to a mammal (e.g., by allowing an orallyadministered compound to be more readily absorbed into the blood), havemore favorable clearance rates or metabolic profiles, or which enhancedelivery of the parent compound to a biological compartment (e.g., thebrain or lymphatic system) relative to the parent species. Preferredprodrugs include derivatives where a group which enhances aqueoussolubility or active transport through the gut membrane is appended tothe structure of formulae (I) and (II).

Pharmaceutically acceptable salts of the compounds of this inventioninclude those derived from pharmaceutically acceptable inorganic andorganic acids and bases. Examples of suitable acid salts includeace-tate, adipate, alginate, aspartate, benzoate, benzene-sulfonate,bisulfate, butyrate, citrate, camphorate, camphorsulfonate,cyclopentanepropionate, digluconate, dodecylsulfate, ethanesulfonate,formate, fumarate, glucoheptanoate, glycerophosphate, glycolate,hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide,hydroiodide, 2-hydroxyethanesulfonate, lactate, maleate, malonate,methanesulfonate, 2-naphtha-lenesulfonate, nicotinate, nitrate, oxalate,palmoate, pectinate, persulfate, 3-phenylpropionate, phosphate, picrate,pivalate, propionate, salicylate, succinate, sulfate, tartrate,thiocyanate, tosylate and undecanoate. Other acids, such as oxalic,while not in themselves pharmaceutically acceptable, may be employed inthe preparation of salts useful as intermediates in obtaining thecompounds of the invention and their pharmaceutically acceptable acidaddition salts.

Salts derived from appropriate bases include alkali metal (e.g., sodiumand potassium), alkaline earth metal (e.g., magnesium), ammonium andN—(C₁₋₄ alkyl)₄ ⁺+ salts. This invention also envisions thequaternization of any basic nitrogen-containing groups of the compoundsdisclosed herein. Water or oil-soluble or dispersible products may beobtained by such quaternization.

In general, compounds of formulae (I) and (II) are obtained via methodsillustrated in Examples 1-8. As can be appreciated by the skilledartisan however the synthetic schemes set forth herein are not intendedto comprise a comprehensive list of all means by which the compoundsdescribed and claimed in this application may be synthesized. Furthermethods will be evident to those of ordinary skill in the art.Additionally, the various synthetic steps described above may beperformed in an alternate sequence or order to give the desiredcompounds.

Without being bound by theory, we believe that the compounds of thisinvention interact either covalently or noncovalently with the activesite of the HCV NS3 protease and other serine proteases, inhibiting theability of such an enzyme to cleave natural or synthetic substrates.Noncovalent interactions are advantageous in that they impart relativelygreater specificity of inhibition and will not inhibit other undesirabletargets, e.g. cysteine proteases. These compounds will therefore have agreater therapeutic index when administered to mammals than covalentprotease inhibitors, which can interact with a wide range of proteasesand cause undesirable toxic effects. In contrast, covalent interactionsare advantageous in that they impart greater inhibitory potency allowinglower doses to be administered and thus ameliorating any lack ofspecificity problems.

The novel compounds of the present invention are excellent inhibitors ofproteases, particularly serine proteases, and more particularly HCV NS3proteases. Accordingly, these compounds are capable of targeting andinhibiting proteases, particularly serine proteases, and moreparticularly HCV NS3 proteases. As such, these compounds interfere withthe life cycle of viruses, including HCV, and are thus useful asantiviral agents. Inhibition can be measured by various methods such asthe methods of Example 11.

The term “antiviral agent” refers to a compound or drug which possessesviral inhibitory activity. Such agents include reverse transcriptaseinhibitors (including nucleoside and non-nucleoside analogs) andprotease inhibitors. Preferably the protease inhibitor is a HCV proteaseinhibitor.

The term “treating” as used herein refers to the alleviation of symptomsof a particular disorder in a patient or the improvement of anascertainable measurement associated with a particular disorder. As usedherein, the term “patient” refers to a mammal, including a human.

Thus, according to another embodiment this invention providespharmaceutical compositions comprising a compound of formula (I) or (II)or a pharmaceutically acceptable salt thereof; an additional agentselected from, but not exclusively, an immunomodulatory agent, such asα-, β-, or γ-interferon; other antiviral agents, such as ribavarin oramantadine; other inhibitors of HCV protease; inhibitors of othertargets in the HCV life cycle such as helicase, polymerase, ormetalloprotease; or combinations thereof and any pharmaceuticallyacceptable carrier, adjuvant or vehicle. An alternate embodimentprovides compositions comprising a compound of formula (I) or (II) or apharmaceutically acceptable salt thereof; and a pharmaceuticallyacceptable carrier, adjuvant or vehicle. Such composition may optionallycomprise an additional agent selected from an immunomodulatory agent,such as α-, β-, or γ-interferon; other antiviral agents, such asribavarin; other inhibitors of HCV protease; inhibitors of HCV helicase;or combinations thereof.

The term “pharmaceutically acceptable carrier or adjuvant” refers to acarrier or adjuvant that may be administered to a patient, together witha compound of this invention, and which does not destroy thepharmacological activity thereof and is nontoxic when administered indoses sufficient to deliver a therapeutic amount of the compound.

Pharmaceutically acceptable carriers, adjuvants and vehicles that may beused in the pharmaceutical compositions of this invention include, butare not limited to, ion exchangers, alumina, aluminum stearate,lecithin, self-emulsifying drug delivery systems (SEDDS) such asdα-tocopherol, polyethyleneglycol 1000 succinate, surfactants used inpharmaceutical dosage forms such as Tweens or other similar polymericdelivery matrices, serum proteins, such as human serum albumin, buffersubstances such as phosphates, glycine, sorbic acid, potassium sorbate,partial glyceride mixtures of saturated vegetable fatty acids, water,salts or electrolytes, such as protamine sulfate, disodium hydrogenphosphate, potassium hydrogen phosphate, sodium chloride, zinc salts,colloidal silica, magnesium trisilicate, polyvinyl pyrrolidone,cellulose-based substances, polyethylene glycol, sodiumcarboxymethylcellulose, polyacrylates, waxes,polyethylene-polyoxypropylene-block polymers, polyethylene glycol andwool fat. Cyclodextrins such as “α-, β-, and γ-cyclodextrin, orchemically modified derivatives such as hydroxyalkylcyclodextrins,including 2- and 3-hydroxypropyl-β-cyclodextrins, or other solubilizedderivatives may also be advantageously used to enhance delivery ofcompounds of formula (I) or (II).

The pharmaceutical compositions of this invention may be administeredorally, parenterally, by inhalation spray, topically, rectally, nasally,buccally, vaginally or via an implanted reservoir. We prefer oraladministration or administration by injection. The pharmaceuticalcompositions of this invention may contain any conventional non-toxicpharmaceutically-acceptable carriers, adjuvants or vehicles. In somecases, the pH of the formulation may be adjusted with pharmaceuticallyacceptable acids, bases or buffers to enhance the stability of theformulated compound or its delivery form. The term parenteral as usedherein includes subcutaneous, intracutaneous, intravenous,intramuscular, intra-articular, intrasynovial, intrasternal,intrathecal, intralesional and intracranial injection or infusiontechniques.

The pharmaceutical compositions may be in the form of a sterileinjectable preparation, for example, as a sterile injectable aqueous oroleaginous suspension. This suspension may be formulated according totechniques known in the art using suitable dispersing or wetting agents(such as, for example, Tween 80) and suspending agents. The sterileinjectable preparation may also be a sterile injectable solution orsuspension in a non-toxic parenterally-acceptable diluent or solvent,for example, as a solution in 1,3-butanediol. Among the acceptablevehicles and solvents that may be employed are mannitol, water, Ringer'ssolution and isotonic sodium chloride solution. In addition, sterile,fixed oils are conventionally employed as a solvent or suspendingmedium. For this purpose, any bland fixed oil may be employed includingsynthetic mono- or diglycerides. Fatty acids, such as oleic acid and itsglyceride derivatives are useful in the preparation of injectables, asare natural pharmaceutically-acceptable oils, such as olive oil orcastor oil, especially in their polyoxyethylated versions. These oilsolutions or suspensions may also contain a long-chain alcohol diluentor dispersant such as those described in Pharmacopeia Helvetica (Ph.Helv.) or a similar alcohol, or carboxymethyl cellulose or similardispersing agents which are commonly used in the formulation ofpharmaceutically acceptable dosage forms such as emulsions and/orsuspensions. Other commonly used surfactants such as Tweens or Spansand/or other similar emulsifying agents or bioavailability enhancerswhich are commonly used in the manufacture of pharmaceuticallyacceptable solid, liquid, or other dosage forms may also be used for thepurposes of formulation.

The pharmaceutical compositions of this invention may be orallyadministered in any orally acceptable dosage form including, but notlimited to, capsules, tablets, and aqueous suspensions and solutions. Inthe case of tablets for oral use, carriers which are commonly usedinclude lactose and corn starch. Lubricating agents, such as magnesiumstearate, are also typically added. For oral administration in a capsuleform, useful diluents include lactose and dried corn starch. Whenaqueous suspensions are administered orally, the active ingredient iscombined with emulsifying and suspending agents. If desired, certainsweetening and/or flavoring and/or coloring agents may be added.

The pharmaceutical compositions of this invention may also beadministered in the form of suppositories for rectal administration.These compositions can be prepared by mixing a compound of thisinvention with a suitable non-irritating excipient which is solid atroom temperature but liquid at the rectal temperature and therefore willmelt in the rectum to release the active components. Such materialsinclude, but are not limited to, cocoa butter, beeswax and polyethyleneglycols.

Topical administration of the pharmaceutical compositions of thisinvention is especially useful when the desired treatment involves areasor organs readily accessible by topical application. For applicationtopically to the skin, the pharmaceutical composition should beformulated with a suitable ointment containing the active componentssuspended or dissolved in a carrier. Carriers for topical administrationof the compounds of this invention include, but are not limited to,mineral oil, liquid petroleum, white petroleum, propylene glycol,polyoxyethylene polyoxypropylene compound, emulsifying wax and water.Alternatively, the pharmaceutical composition can be formulated with asuitable lotion or cream containing the active compound suspended ordissolved in a carrier. Suitable carriers include, but are not limitedto, mineral oil, sorbitan monostearate, polysorbate 60, cetyl esterswax, cetearyl alcohol, 2-octyldodecanol, benzyl alcohol and water. Thepharmaceutical compositions of this invention may also be topicallyapplied to the lower intestinal tract by rectal suppository formulationor in a suitable enema formulation. Topically-transdermal patches arealso included in this invention.

The pharmaceutical compositions of this invention may be administered bynasal aerosol or inhalation. Such compositions are prepared according totechniques well-known in the art of pharmaceutical formulation and maybe prepared as solutions in saline, employing benzyl alcohol or othersuitable preservatives, absorption promoters to enhance bioavailability,fluorocarbons, and/or other solubilizing or dispersing agents known inthe art.

Dosage levels of between about 0.01 and about 100 mg/kg body weight perday, preferably between about 0.5 and about 75 mg/kg body weight per dayof the protease inhibitor compounds described herein are useful in amonotherapy for the prevention and treatment of antiviral, particularlyanti-HCV mediated disease. Typically, the pharmaceutical compositions ofthis invention will be administered from about 1 to about 5 times perday or alternatively, as a continuous infusion. Such administration canbe used as a chronic or acute therapy. The amount of active ingredientthat may be combined with the carrier materials to produce a singledosage form will vary depending upon the host treated and the particularmode of administration. A typical preparation will contain from about 5%to about 95% active compound (w/w). Preferably, such preparationscontain from about 20% to about 80% active compound.

When the compositions of this invention comprise a combination of acompound of formula (I) or (II) and one or more additional therapeuticor prophylactic agents, both the compound and the additional agentshould be present at dosage levels of between about 10 to 100%, and morepreferably between about 10 to 80% of the dosage normally administeredin a monotherapy regimen.

According to one embodiment, the pharmaceutical compositions of thisinvention comprise an additional immunomodulatory agent. Examples ofadditional immunomodulatory agents include, but are not limited to, α-,β-, and γ-interferons.

According to an alternate embodiment, the pharmaceutical compositions ofthis invention may additionally comprise an anti-viral agent. Examplesof anti-viral agents include ribavirin and amantadine.

According to another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise otherinhibitors of HCV protease.

According to yet another alternate embodiment, the pharmaceuticalcompositions of this invention may additionally comprise an inhibitor ofother targets in the HCV life cycle, such as helicase, polymerase, ormetalloprotease.

Upon improvement of a patient's condition, a maintenance dose of acompound, composition or combination of this invention may beadministered, if necessary. Subsequently, the dosage or frequency ofadministration, or both, may be reduced, as a function of the symptoms,to a level at which the improved condition is retained. When thesymptoms have been alleviated to the desired level, treatment shouldcease. Patients may, however, require intermittent treatment on along-term basis upon any recurrence of disease symptoms.

As the skilled artisan will appreciate, lower or higher doses than thoserecited above may be required. Specific dosage and treatment regimensfor any particular patient will depend upon a variety of factors,including the activity of the specific compound employed, the age, bodyweight, general health status, sex, diet, time of administration, rateof excretion, drug combination, the severity and course of theinfection, the patient's disposition to the infection and the judgmentof the treating physician.

When these compounds or their pharmaceutically acceptable salts areformulated together with a pharmaceutically acceptable carrier, theresulting composition may be administered in vivo to mammals, such asman, to inhibit serine proteases, particularly HCV NS3 protease or totreat or prevent viral infection, particularly HCV virus infection. Suchtreatment may also be achieved using the compounds of this invention incombination with agents which include, but are not limited to:immunomodulatory agents, such as α-, β-, or γ-interferons; otherantiviral agents such as ribavirin, amantadine; other inhibitors of HCVNS3 protease; inhibitors of other targets in the HCV life cycle such ashelicase, polymerase, metalloprotease, or internal ribosome entry; orcombinations thereof. These additional agents may be combined with thecompounds of this invention to create a single dosage form.

Alternatively these additional agents may be separately administered toa mammal as part of a multiple dosage form.

Accordingly, another embodiment of this invention provides methods ofinhibiting serine protease activity in mammals by administering acompound of formula (I) or (II), wherein the substituents are as definedabove. Preferably, the serine protease is HCV NS3.

In an alternate embodiment, the invention provides methods of inhibitingHCV or HCV NS3 activity in a mammal comprising the step of administeringto said mammal, a compound of formula (I) or (II), wherein thesubstituents are as defined above.

In an alternate embodiment, this invention provides methods ofdecreasing serine protease activity in a mammal comprising the step ofadministrating to said mammal any of the pharmaceutical compositions andcombinations described above. If the pharmaceutical compositioncomprises only a compound of this invention as the active component,such methods may additionally comprise the step of administering to saidmammal an agent selected from an immunomodulatory agent, an antiviralagent, a HCV protease inhibitor, or an inhibitor of other targets in theHCV life cycle. Such additional agent may be administered to the mammalprior to, concurrently with, or following the administration of the HCVinhibitor composition.

In a preferred embodiment, these methods are useful in decreasing HCVNS3 protease activity in a mammal. If the pharmaceutical compositioncomprises only a compound of this invention as the active component,such methods may additionally comprise the step of administering to saidmammal an agent selected from an immunomodulatory agent, an antiviralagent, a HCV protease inhibitor, or an inhibitor of other targets in theHCV life cycle such as helicase, polymerase, or metalloprotease. Suchadditional agent may be administered to the mammal prior to,concurrently with, or following the administration of the compositionsof this invention.

In an alternate preferred embodiment, these methods are useful forinhibiting viral replication in a mammal. Such methods are useful intreating or preventing, for example, viral diseases, such as HCV. If thepharmaceutical composition comprises only a compound of this inventionas the active component, such methods may additionally comprise the stepof administering to said mammal an agent selected from animmunomodulatory agent, an antiviral agent, a HCV protease inhibitor, oran inhibitor of other targets in the HCV life cycle. Such additionalagent may be administered to the mammal prior to, concurrently with, orfollowing the administration of the composition according to thisinvention.

The compounds set forth herein may also be used as laboratory reagents.The compounds of this invention may also be used to treat or preventviral contamination of materials and therefore reduce the risk of viralinfection of laboratory or medical personnel or patients who come incontact with such materials. These materials include, but are notlimited to, biological materials, such as blood, tissue, etc; surgicalinstruments and garments; laboratory instruments and garments; and bloodcollection apparatuses and materials.

In order that this invention be more fully understood, the followingexamples are set forth. These examples are for the purpose ofillustration only and are not to be construed as limiting the scope ofthe invention in any way.

General Materials and Methods

A general synthetic methodology for preparing compounds of thisinvention is provided in Example 1. More specific methodologies forpreparing compounds of this invention, including compounds I-198, areprovided in Examples 2-9.

The HPLC data reported in Tables 1-7 is expressed in terms of solventgradient, retention time, and % purity. Deionized water was used in eachmethod.

The correct (M+H)⁺ and/or (M+Na)⁺ molecular ions for all compounds wereobtained by either matrix-assisted laser desorption mass spectrometry(Kratos MALDI I) or by electro spray mass spectrometry (MICROMASS QuatroII).

EXAMPLE 1

Numerous amino acids for use in the synthesis of peptidyl andpeptidomimetic compounds of this invention may be purchased commerciallyfrom, for instance, Sigma Chemical Company or Bachem Feinchemikalien AG(Switzerland). Amino acids that are not commercially available can bemade by known synthetic routes (“Kinetic Resolution of Unnatural andRarely Occurring Amino Acids: Enantioselective Hydrolysis of N-AcylAmino Acids Catalyzed by Acylase I”, Chenault, H. K. et. al., J. Am.Chem. Soc. 111, 6354-6364 (1989) and references cited therein;“Synthesis of β-β-Unsaturated Amino Acids by the Strecker Reaction,Greenlee, W. J., J. Org. Chem. 49, 2632-2634 (1984); “RecentStereoselective Synthetic Approaches to Beta-amino Acids”, Cole, D.Tetrahedron 50: 9517 (1994); “The Chemistry of Cyclic Alpha IminoAcids”, Mauger, A. B; Volume 4 of “Chemistry and Biochemistry of AminoAcids, Peptides, and Proteins”, Weinstein, B. editor, Marcel Dekker(1977); “Recent Progress in the Synthesis and Reactions of SubstitutedPiperidines”, Org. Prep. Procedure Int. 24, 585-621 (1992), all of whichare incorporated herein by reference).

Certain compounds of formula (I) or (II) may be synthesized from aminoacids by procedures which are well known in the art of peptide andorganic chemical synthesis. Examples of such syntheses are generally setforth in Bodanszky and Bodanszky, “The Practice of Peptide Synthesis”,Springer-Verlag, Berlin, Germany (1984), “The Peptides”, Gross andMeinhofer, eds; Academic Press, 1979, Vols. I-III, and Stewart, J. M.and Young, J. D., “Solid Phase Peptide Synthesis, Second Edition”,Pierce Chemical Company, Rockford, Ill. (1984); and “Recent Advances inthe Generation of Molecular Diversity”, Moos, W. H., Green, G. D. andPavia, M. R. in “Annual Reports in Medicinal Chemistry, Vol. 28” pp.315-324; Bristol, J. A., ed.; Academic Press, San Diego, Calif. (1993),all of which are incorporated herein by reference.

Typically, for solution phase synthesis of peptides, the α-amine of theamino acid to be coupled is protected by a urethane such as Boc, Cbz,Fmoc or Alloc while the free carboxyl is activated by reaction with acarbodiimide such as DCC, EDC, or DIC, optionally in the presence of acatalyst such as HOBT, HOAt, HOSu, or DMAP. Other methods, which proceedthrough the intermediacy of activated esters, acid halides,enzyme-activated amino acids and anhydrides including phosphoniumreagents such as BOP, Py-BOP, N-carboxy-anhydrides, symmetricalanhydrides, mixed carbonic anhydrides, carbonic-phosphinic andcarbonic-phosphoric anhydrides, are also suitable. After the peptide hasbeen formed, protecting groups may be removed by methods described inthe references listed above, such as by hydrogenation in the presence ofa palladium, platinum or rhodium catalyst, treatment with sodium inliquid ammonia, hydrochloric, hydrofluoric, hydrobromic, formic,trifluoromethanesulfonic, or trifluoroacetic acid, secondary amines,fluoride ion, trimethylsilyl halides including bromide and iodide, oralkali. Automation of the synthetic process, using techniques such asthose set forth above, can be accomplished by use of commerciallyavailable instrumentation, including but not limited to the AdvancedChemtech 357 FBS and 496 MOS; Tecan CombiTec, and Applied Biosystems433A among others. Specific application of these methods and theirequivalents, depending upon the target compound, will be apparent tothose skilled in the art. Modifications of chemical processes and choiceof instrumentation is within the skill of the ordinary practitioner.

EXAMPLE 2

Compounds 1-26 (Table 1) were prepared as described in scheme 1.

Synthesis of 301-306

Step A. Synthesis of 301. 4-Methyl Benzhydrylamine resin (1.05 mmol/g,20.0 g) was placed in a sintered glass funnel and washed withdimethylformamide (3×75 mL), 10% (v/v) diisopropylethylamine (DIEA) indimethylformamide (2×75 mL) and finally with dimethylformamide (4×75mL). Sufficient dimethylformamide was added to the resin to obtain aslurry followed by 300 (8.0 g, 20.8 mmol, prepared from (2S)2-(t-Butyloxycarbonylamino)-butyraldehyde according to A. M. Murphy et.al. J. Am. Chem. Soc., 114, 3156-3157 (1992)), 1-hydroxybenzotriazolehydrate (HOBT-H₂O; 3.22 g, 21.0 mmol),O-benzotriazole-N,N,N′,N′-tetramethyluronium hexafluorophosphate (HBTU;8.0 g, 21.0 mmol), and DIEA (11.0 mL, 63 mmol). The reaction mixture wasagitated overnight at room temperature using a wrist arm shaker. Theresin was isolated on a sintered glass funnel by suction filtration andwashed with dimethylformamide (3×75 mL). Unreacted amine groups werethen capped by reacting the resin with 20% (v/v) aceticanhydride/dimethylformamide (2×50 mL) directly in the funnel (10min/wash). The resin was washed with dimethylformamide (3×75 mL) anddichloromethane (3×75 mL) prior to drying overnight in vacuo to yield300a (26.3 g, 81% yield).

The t-Boc protecting group was removed from resin 300a using theAdvanced ChemTech 396 Multiple Peptide synthesizer by the followingprocedure. Resin 300a (0.05 mmol) was swelled by washing withdichloromethane (3×1 mL) followed by cleavage of the t-Boc protectinggroup with 50% (v/v) TFA/dichloromethane (1.0 mL) for 10 min (withshaking) followed by fresh reagent (1 mL) for 30 min. The resin was thenwashed with dichloromethane (3×1 ml), followed by DMF (3×1 mL), then 10%DIEA/dimethylformamide (v/v) (2×1 mL), and finally withN-methylpyrrolidone (3×1 mL) to yield resin 301.

Step B. Synthesis of 303. This compound was prepared from resin 301(0.05 mmol) using an Advanced ChemTech 396 Multiple Peptide synthesizer.Resin 301 was acylated with a solution of 0.4M 302 and 0.4M HOBT inN-methylpyrrolidone (0.5 mL), a solution of 0.4M HBTU inN-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA inN-methylpyrrolidone (0.35 mL) and the reaction was shaken for 4 hr atroom temperature. The coupling reaction was repeated. The resin was thenwashed with dimethylformamide (3×1 ml), followed by dichloromethane (3×1mL) to yield resin 303.

Step C. Synthesis of 305. The synthesis of the resin-bound compound wascompleted using an Advanced ChemTech 396 Multiple Peptide synthesizer.Resin 303 was washed with dichloromethane (3×1 mL) followed by cleavageof the t-Boc protecting group with 50% (v/v) TFA/dichloromethane (1.0mL) for 10 min (with shaking) followed by fresh reagent (1 mL) for 30min. The resin was then washed with dichloromethane (3×1 ml), followedby DMF (3×1 mL), then 10% DIEA/dimethylformamide (v/v) (2×1 mL), andfinally with N-methypyrrolidone (3×1 mL) to yield resin 304. This resinwas then acylated with a solution of 0.4M Fmoc-Valine and 0.4M HOBT inN-methylpyrrolidone (0.5 mL), a solution of 0.4M HBTU inN-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA inN-methylpyrrolidone (0.35 mL) and the reaction was shaken for 4 hr atroom temperature. The coupling reaction was repeated. The automatedcycles consisted of: (1) a resin wash with dimethylformamide (3×1 mL);(2) deprotection with 25% (v/v) piperidine in dimethylformamide (1 mL)for 3 min followed by fresh reagent (1 mL) for 10 min.; (3) a resin washwith dimethylformamide (3×1 mL) and N-methylpyrrolidone (3×1 mL) priorto coupling as described above. Fmoc-Valine and Pyrazine-2-carboxylicacid were coupled in this manner.

Step D. Synthesis of 306. Prior to cleavage, the resin was washed with1:1 dichloromethane/methanol (3×1 mL) and then dried in vacuo. Thealdehyde was cleaved from the resin by treatment with either 95% TFA/5%H₂O (v/v, 1.5 mL) for 30 min at room temperature or by treatment withtetrahydrofuran/30% formalin/1N HCl 9:1:1 (v:v:v) for 1 hr at roomtemperature. After washing the resin with cleavage reagent (1 mL), thecombined filtrates were diluted with water and lyophilized to obtaincrude 306 as a white powder. The compound was purified by semi-prepRP-HPLC with a Waters DeltaPak 300 Å C18 column (15μ, 30×300 mm) elutingwith a linear acetonitrile gradient containing 0.1% TFA (v/v) over 45min at 20 mL/min. Fractions containing the desired product were pooledand lyophilized to provide 306.

EXAMPLE 3

Compounds 27-29 (Table 1) were prepared as described in scheme 2.

Step A. Synthesis of 301. See Step A, Scheme 1 methodology.

Step B. Synthesis of 308. Resin 301 (6.0 g, 0.65 mmol/g, 3.9 mmol) wasswelled in a sintered glass funnel by washing with dichloromethane (3×50mL). The Boc protecting group was then cleaved with 50% (v/v)TFA/dichloromethane (50 mL) for 10 min (intermittent stirring) and thenfor 30 min with fresh reagent (50 ml). The resin was then washed withdichloromethane (3×50 ml), dimethylformamide (2×50 mL), 10%DIEA/dimethylformamide (v/v) (2×50 mL), and finally N-methylpyrrolidone(3×50 mL). After transferring the resin to a 100 mL flask,N-methylpyrrolidone was added to obtain a slurry followed by 307 (2.83g, 8.0 mmol), HOBT.H₂O (1.22 g, 8.0 mmol), HBTU (3.03 g, 8.0 mmol) andDIEA (4.2 mL, 24 mmol). The reaction mixture was agitated overnight atroom temperature using a wrist arm shaker. The resin work-up and cappingwith 20% (v/v) acetic anhydride in dimethylformamide were performed asdescribed for 301 to yield 308 (6.86 g, quantitative yield).

Step C. Synthesis of 309. This compound was prepared from resin 308(0.15 mmol) using a Tecan CombiTec synthesizer. Resin 308 (0.076 mmol)was washed with dimethylformamide (3×2 mL), deprotected with 25% (v/v)piperidine in dimethylformamide (2.5 mL) for 5 min followed by freshreagent (2 mL) for 20 min. The resin was washed with dimethylformamide(3×2.5 mL) and N-methylpyrrolidone (3×2.5 mL) prior to acylation with asolution of 0.4M Fmoc-Valine and 0.4M HOBT in N-methylpyrrolidone (0.8mL), a solution of 0.4M HBTU in N-methylpyrrolidone (0.8 mL) and asolution of 1.6M DIEA in N-methylpyrrolidone (0.6 mL) and the reactionwas shaken for 8 hr at room temperature. The coupling reaction wasrepeated. The deprotection and coupling procedures were repeated to addthe second Valine residue and to add the final pyrazine-2-carboxylicacid residue. The resin was then washed with dichloromethane (3×2.5 ml)to yield resin 309.

Step D. Synthesis of 310. To resin 309 was added 1:1pyridine/dichloromethane (v/v) (1 mL), 0.8M dimethylaminopyridine indimethylformamide (0.2 mL), and a solution of 0.2M Z-COCl indichloromethane (1.5 mL) and the reaction was shaken for 8 hr at roomtemperature. The acylation reaction was repeated. The resin was washedwith dichloromethane (3×2.5 mL), dimethylformamide (3×2.5 mL),dichloromethane (3×2.5 mL), and finally with 1:1dichloromethane/methanol (3×2.5 mL) to yield resin 310.

Step E. Synthesis of 311. Prior to cleavage, the resin was washed with1:1 dichloromethane/methanol (3×1 mL) and then dried in vacuo. Thealdehyde was cleaved from the resin by treatment withtetrahydrofuran/formalin/acetic acid/1N HCl 5:1:1:0.1 (v:v:v:v) for 1 hrat room temperature. After washing the resin with cleavage reagent (1mL), the combined filtrates were diluted with water and lyophilized toobtain crude 311 as a white powder. The compound was purified bysemi-prep RP-HPLC with a Waters DeltaPak 300 Å C18 column (15μ, 30×300mm) eluting with a linear acetonitrile gradient containing 0.1% TFA(v/v) over 45 min at 20 mL/min. Fractions containing the desired productwere pooled and lyophilized to provide 311.

EXAMPLE 4

Compounds 30-56 (Table 1) were prepared as described in scheme 3.

Step A. Synthesis of 301. See Step A, Scheme 1 methodology.

Step B. Synthesis of 308. See Step B, Scheme 2 methodology.

Step C. Synthesis of 312. This compound was prepared from resin 308(0.15 mmol) using a Tecan CombiTec synthesizer. Resin 308 was washedwith toluene (3×2.5 mL) and then suspended in toluene (1.0 mL). To thiswas added either a solution of 0.8M R₃ δ-isocyanate in toluene (1.0 mL)followed by 0.8M DIEA in toluene (1.0 mL) or a solution of 0.8M R₃δ-carboxylic acid with 0.8M DIEA in toluene (1.0 mL) followed by 0.8Mdiphenylphosphorylazide in toluene (1.0 mL). The reaction was shaken for8 hr at 55° C. The resin was then washed with toluene (3×2.5 ml) anddimethylformamide (4×2.5 mL) to yield resin 312.

Step D. Synthesis of 313. See Step D, Scheme 2 methodology.

Step E. Synthesis of 314. See Step E, Scheme 2 methodology.

EXAMPLE 5

Compounds 57-70 (Table 1) were prepared as described in scheme 4.

Step A. Synthesis of 301. See Step A, Scheme 1 methodology.

Step B. Synthesis of 316. This compound was prepared from resin 301(0.05 mmol) using an Advanced ChemTech 396 Multiple Peptide synthesizer.Resin 301 was acylated with a solution of 0.4M 315 and 0.4M HOBT inN-methylpyrrolidone (0.5 mL), a solution of 0.4M HBTU inN-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA inN-methylpyrrolidone (0.35 mL) and the reaction was shaken for 4 hr atroom temperature. The coupling reaction was repeated. The resin was thenwashed with dimethylformamide (3×1 ml), followed by dichloromethane (3×1mL). The Boc protecting group was then cleaved with 50% (v/v)TFA/dichloromethane (1.0 mL) for 10 min with vortexing and then for 30min with fresh reagent (1.0 ml). The resin was then washed withdichloromethane (3×1.0 ml), dimethylformamide (2×1.0 mL), 10%DIEA/dimethylformamide (v/v) (2×1.0 mL), dimethylformamide (3×1.0 ml),and finally dichloromethane (3×1.0 mL) to yield 316.

Step C. Synthesis of 317a. Resin 316 was acylated with a solution of0.4M Z-CO₂H and 0.4M HOBT in N-methylpyrrolidone (0.5 mL), a solution of0.4M HBTU in N-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA inN-methylpyrrolidone (0.35 mL) and the reaction was shaken for 4 hr atroom temperature. The coupling reaction was repeated. The resin was thenwashed with dimethylformamide (3×1 ml) to yield resin 317a.

Step C. Synthesis of 317b. Resin 316 was acylated with 0.5M Z-COCl indimethylformamide (1 mL) and 1.6M DIEA in N-methylpyrrolidone (0.35 mL)for 2 hr at room temperature. The acylation step was repeated. The resinwas washed with dimethylformamide (3×2.5 mL) to yield resin 317b.

Step C. Synthesis of 317c. Resin 316 was reacted with 1.0M Z-sulfonylchloride in dichloromethane (0.5 mL) and 1M pyridine in dichloromethane(0.60 mL) for 4 hr at room temperature The reaction was repeated. Theresin was washed with dichloromethane (3×1.0 mL), and thendimethylformamide (3×1.0 mL) to yield resin 317c.

Step C. Synthesis of 317d. Resin 316 was reacted with 0.5M Z-isocyanatein dimethylformamide (1.2 mL) for 8 hr at room temperature The reactionwas repeated. The resin was washed with dimethylformamide (3×1.0 mL) toyield resin 317d.

Step C. Synthesis of 317e. Resin 316 was reacted with 0.5M Z-CHO indimethylformamide (1.2 mL) in the presence of acetic acid (0.1 mL) andsodium cyanoborohydride (200 mg) for 4 hr at room temperature. Thereaction was repeated. The resin was washed with dimethylformamide(3×1.0 mL) to yield resin 317e.

Step D. Synthesis of 318. The synthesis of the resin-bound compound wascompleted using an Advanced ChemTech 396 Multiple Peptide synthesizer.The automated cycles described in Step C, Scheme 1, were used to addFmoc-Valine, followed by another Fmoc-Valine, and finallypyrazine-2-carboxylic acid.

Step E. Synthesis of 319. See Step E, Scheme 2 methodology.

EXAMPLE 6

Compounds 81-100 and 127-142 (Tables 3 and 4) were prepared as describedin scheme 5.

Step A. Synthesis of 301. See Step A, Scheme 1 methodology.

Step B. Synthesis of 320. The synthesis of the resin-bound compound wasaccomplished using an Advanced ChemTech 396 Multiple Peptide synthesizerstarting with resin 165 (0.05 mmol). The automated cycles described inStep C, Scheme 1, were used to add Fmoc-A¹, followed by Fmoc-valine andfinally a terminal Fmoc-amino acid. The Fmoc group was removed aspreviously described with 25% piperidine/dimethylformamide (v:v) toyield resin 166.

Step C. Synthesis of 321a. Resin 320 was acylated with a solution of0.4M T-CO₂H and 0.4M HOBT in N-methylpyrrolidone (0.5 mL), a solution of0.4M HBTU in N-methylpyrrolidone (0.5 mL) and a solution of 1.6M DIEA inN-methylpyrrolidone (0.35 mL) and the reaction was shaken for 4 hr atroom temperature. The coupling reaction was repeated. The resin was thenwashed with dimethylformamide (3×1 ml), dichloromethane (3×1.0 mL), and1:1 dichloromethane/methanol (v/v) (3×1 mL) to yield resin 321a.

Step C. Synthesis of 321b. Resin 320 was acylated with 0.5M T-COCl indimethylformamide (1 mL) and 1.6M DIEA in N-methylpyrrolidone (0.35 mL)for 2 hr at room temperature. The acylation step was repeated. The resinwas then washed with dimethylformamide (3×1 ml), dichloromethane (3×1.0mL), and 1:1 dichloromethane/methanol (v/v) (3×1 mL) to yield resin321b.

Step C. Synthesis of 321c. Resin 320 was reacted with 1.0M T-sulfonylchloride in dichloromethane (0.5 mL) and 1M pyridine in dichloromethane(0.60 mL) for 4 hr at room temperature The reaction was repeated. Theresin was then washed with dimethylformamide (3×1.0 ml), dichloromethane(3×1.0 mL), and 1:1 dichloromethane/methanol (v/v) (3×1.0 mL) to yieldresin 303c.

Step C. Synthesis of 321d. Resin 320 was reacted with 0.5M T-isocyanatein dimethylformamide (1.2 mL) for 8 hr at room temperature The reactionwas repeated. The resin was then washed with dimethylformamide (3×1.0ml), dichloromethane (3×1.0 mL), and 1:1 dichloromethane/methanol (v/v)(3×1.0 mL) to yield resin 321d.

Step D. Synthesis of 322. The aldehyde was cleaved from the resin andglobally deprotected by treatment with 95% TFA/5% H₂O (v/v, 1.5 mL) for45 min at room temperature. After washing the resin with fresh cleavagereagent (1 mL), the combined filtrates were added to cold 1:1ether:pentane (12 mL) and the resulting precipitate was isolated bycentrifugation and decantation. The resulting pellet was dissolved in10% acetonitrile/90% H₂O/0.1% TFA (15 mL) and lyophilized to obtaincrude 322 as a white powder. The compound was purified by semi-prepRP-HPLC with a Waters DeltaPak 300 Å C18 column (15μ, 30×300 mm) elutingwith a linear acetonitrile gradient containing 0.1% TFA (v/v) over 45min at 20 mL/min. Fractions containing the desired product were pooledand lyophilized to provide 322.

EXAMPLE 7

Compounds 143-197 (Table 6) were prepared as described in scheme 6.

Step A. Synthesis of 301. See Step A, Scheme 1 methodology.

Step B. Synthesis of 326. This compound was prepared from resin 301(0.50 mmol) using an Applied Biosystems Model 433A Peptide synthesizer.N^(α)-Fmoc-protected amino acids were added sequentially to resin 301with standard coupling cycles using HBTU with HOBt as coupling agents inN-methylpyrrolidinone to yield resin 326.

Step C. Synthesis of 327a. The synthesis was completed using an AdvancedChemTech 396 Multiple Peptide

Synthesizer. Resin 326 (0.05 mmol) was deprotected with 25% (v/v)piperidine in dimethylformamide (1 mL) for 3 min followed by freshreagent (1 mL) for 10 min. The resin was washed with dimethylformamide(3×1 mL) and N-methylpyrrolidone (3×1 mL). The resin was acylated with asolution of 0.4M T-CO₂H and 0.4M HOBT in N-methylpyrrolidone (0.5 mL), asolution of 0.4M HBTU in N-methylpyrrolidone (0.5 mL) and a solution of1.6M DIEA in N-methylpyrrolidone (0.35 mL) and the reaction was shakenfor 4 hr at room temperature. The coupling reaction was repeated. Theresin was then washed with dimethylformamide (3×1 ml), dichloromethane(3×1.0 mL), and 1:1 dichloromethane/methanol (v/v) (3×1 mL) to yieldresin 327a.

Step C. Synthesis of 327b. The synthesis was completed using an AdvancedChemTech 396 Multiple Peptide Synthesizer. Resin 326 (0.05 mmol) wasdeprotected with 25% (v/v) piperidine in dimethylformamide (1 mL) for 3min followed by fresh reagent (1 mL) for 10 min. The resin was washedwith dimethylformamide (3×1 mL) and N-methylpyrrolidone (3×1 mL). Theresin was acylated with 0.5M T-COCl in dimethylformamide (1 mL) and 1.6MDIEA in N-methylpyrrolidone (0.35 mL) for 2 hr at room temperature. Theacylation step was repeated. The resin was then washed withdimethylformamide (3×1 ml), dichloromethane (3×1.0 mL), and 1:1dichloromethane/methanol (v/v) (3×1 mL) to yield resin 327b.

Step C. Synthesis of 327c. The synthesis was completed using an AdvancedChemTech 396 Multiple Peptide Synthesizer. Resin 326 (0.05 mmol) wasdeprotected with 25% (v/v) piperidine in dimethylformamide (1 mL) for 3min followed by fresh reagent (1 mL) for 10 min. The resin was washedwith dimethylformamide (3×1 mL) and dichloromethane (3×1 mL). The resinwas reacted with 1.0M T-sulfonyl chloride in dichloromethane (0.5 mL)and 1M pyridine in dichloromethane (0.60 mL) for 4 hr at roomtemperature The reaction was repeated. The resin was then washed withdimethylformamide (3×1.0 ml), dichloromethane (3×1.0 mL), and 1:1dichloromethane/methanol (v/v) (3×1.0 mL) to yield resin 327c.

Step C. Synthesis of 327d. The synthesis was completed using an AdvancedChemTech 396 Multiple Peptide

Synthesizer. Resin 326 (0.05 mmol) was deprotected with 25% (v/v)piperidine in dimethylformamide (1 mL) for 3 min followed by freshreagent (1 mL) for 10 min. The resin was washed with dimethylformamide(3×1 mL). The resin was reacted with 0.5M T-isocyanate indimethylformamide (1.2 mL) for 8 hr at room temperature The reaction wasrepeated. The resin was then washed with dimethylformamide (3×1.0 ml),dichloromethane (3×1.0 mL), and 1:1 dichloromethane/methanol (v/v)(3×1.0 mL) to yield resin 327d.

Step D. Synthesis of 328. See Step D, Scheme 1 methodology.

EXAMPLE 8

Compounds 79-80 and 101-123 (Tables 2, 3 and 4) were prepared asdescribed in scheme 7.

Step A. Synthesis of 330. 2-Chlorochlorotrityl resin (2.2 mmol/g, 1.69g) was reacted with 329 (0.385 g, 1.1 mmol, prepared according to S. L.Harbeson et. al. J. Med. Chem., 37, 2918 (1994)) in dichloromethane inthe presence of DIEA (0.47 mL, 2.7 mmol) at room temperature for 1 hour.The reaction was quenched by the addition of methanol and the resin wasisolated on a sintered glass funnel by suction filtration and washedwith dichloromethane (3×25 mL). The resin was dried overnight in vacuoto yield 330 (1.92 g, 0.49 meq/g).

Step B. Synthesis of 332. The synthesis of the resin-bound compound wasaccomplished using an Applied Biosystems Model 433A Peptide synthesizerstarting with resin 330 (0.74 mmol). The automated cycles described inStep C, Scheme 1, were used to add Fmoc-A¹, followed by Fmoc-A² andFmoc-A³. The Fmoc group was removed as previously described with 25%piperidine/dimethylformamide (v:v) to yield resin 332.

Step C. Synthesis of 333. Prior to cleavage, the resin was washed with1:1 dichloromethane/methanol (3×1 mL) and then dried in vacuo. Thepeptide was cleaved from the resin by treatment with aceticacid:trifluoroethanol:dichloromethane (1:1:3) for 1 hr at roomtemperature. After washing the resin with dichloromethane, the combinedfiltrates were concentrated in vacuo to obtain crude 333 as a whitepowder (0.48 g, 76%).

Step D. Synthesis of 335. Compound 333 (0.05 g, 0.058 mmol) wasdissolved in dimethylacetamide (1 mL) and to this was added DIEA (0.17mmol), the appropriate amine (0.20 mmol), and PyBrop (0.12 mmol). Thereaction was stirred for 2 hr at 70° C. The reaction was then dilutedinto H₂O (8 mL) followed by centrifugation to obtain the precipitatethat was dried in vacuo to obtain crude 334, which was then oxidizeddirectly to compound 335. Crude 334 was dissolved in N-methylpyrrolidone(3 mL) and reacted with Dess-Martin periodinane (110 mg, 0.26 mmol) atroom temperature over night. Saturated aqueous sodium bicarbonate (5 mL)and 10% (w:v) aqueous sodium thiosulfate (5 mL) were added to thereaction and stirred prior to addition of H₂O (40 mL). The precipitatewas isolated by centrifugation and the solid was dried in vacuo. Whenrequired, acid labile protecting groups were removed by treatment with1:1 trifluoroacetic acid:dichloromethane at room temperature for 30 min.The solvent was removed in vacuo and the crude compound was purified bysemi-prep RP-HPLC with a Waters DeltaPak 300 Å C18 column (15μ, 30×300mm) eluting with a linear acetonitrile gradient containing 0.1% TFA(v/v) over 45 min at 20 mL/min. Fractions containing the desired productwere pooled and lyophilized to provide 335.

EXAMPLE 9

Compounds 71-78 and 124-126 were prepared from the appropriate protectedpeptide acids. Protected peptide acids were prepared as previouslydescribed in Scheme 7 using 2-Chloro-chlorotrityl resin. These peptideacids were then coupled to one of the following groups using standardsolution phase peptide coupling methodologies. The references forpreparation of these groups are also given.

J. Oleksyszyn et. al., Synthesis, 985-986 (1979)

S. Elgendy et. al., Tetrahedron, 50, 3803-3812 (1994)

M. R. Angelestro et. al., Tetrahedron Letters, 33, 3265-3268 (1992)

T. T. Curran, J. Organic Chemistry, 58, 6360-6363 (1993)

E. Edwards, et. al., J. Medicinal Chemistry, 38, 3972-3982 (1995).

When required, the products obtained were oxidized to the ketones usingDess Martin Periodinane as described for Scheme 7. When required, acidlabile protecting groups were removed by treatment with 1:1trifluoroacetic acid:dichloromethane at room temperature for 30 min. Thesolvent was removed in vacuo and the crude compound was purified bysemi-prep RP-HPLC with a Waters DeltaPak 300 Å C18 column (15μ, 30×300mm) eluting with a linear acetonitrile gradient containing 0.1% TFA(v/v) over 45 min at 20 mL/min. Fractions containing the desired productwere pooled and lyophilized to provide the final products 71-78 and124-126.

EXAMPLE 10

Compound 198 was prepared by modification of the general methodologydescribed in Example 1.

TABLE 1 Structures and analytical data - compounds 1-70.

Z X Y MS Data HPLC 1

O CH₂ ND 40-80%B; 5.484 min.; 6.580 min; 75:25 fast:slow 2

O CH₂ (M + Na) = 693.2 40-80%B; 5.376 min; 95% 3

O CH₂ (M + H) = 664.0 (M + Na) = 685.2 20-60%B; 8.527 min.; 100% 4

O CH₂ (M + Na) = 714.3 20-60%B; 8.885 min.; 100% 5

O CH₂ (M + H) = 682.9, (M + Na) = 704.0 20-60%B; 7.541 min.; 95.6% 6

O CH₂ (M + H) = 644.0, (M + Na) = 664.0 20-60%B; 7.822 min.; 100% 7

O CH₂ (M + H) = 746.7, (M + Na) = 765.7 40-80%B; 4.228 min.; 92% 8

O CH₂ (M + H) = 671.5, (M + Na) = 694.0 20-60%B; 8.554 min.; 98% 9

O CH₂ (M + Na) = 700.9 40-80%B; 4.688 min.; 100% 10

O CH₂ (M + Na) = 686.3 40-80%B; 4.630 min.; 94% 11

O CH₂ (M + H) = 671.1, (M + Na) = 693.2 40-80%B; 5.323 min.; 6.435 min.;88:12, fast:slow 12

O CH₂ (M + H) = 613.7, (M + Na) = 636.2 20-60%B; 5.696 min.; 100% 13

O CH₂ (M + Na) = 695.3 20-60%B; 9.046 min.; 100% 14

O CH₂ (M + Na) = 714.9 20-60%B; 7.729 min.; 100% 15

O CH₂ (M + Na) = 642.72 20-60%B; 7.133 min.; 100% 16

O CH₂ (M + Na) = 685.6 20-60%B; 10.177 min.; 100% 17

O CH₂ (M + Na) = 685.6 20-60%B; 10.265 min.; 100% 18

O CH₂ (M + Na) = 700.9 20-60%B; 10.696 min.; 100% 19

O CH₂ (M + Na) = 709.4 30-70%B; 9.216 min.; 100% 20

O CH₂ (M + Na) = 667.3 20-60%B; 10.225 min; 100% 21

O CH₂ (M + Na) = 641.8 20-60%B; 7.15 min.; 100% 22

O CH₂ (M + Na) = 653.1 20-60%B; 8.822 min.; 100% 23

O CH₂ (M + Na) = 707.3 20-60%B; 11.362 min.; 100% 24

O CH₂ (M + Na) = 733.9 20-60%B; 10.964 min.; 100% 25

O CH₂ (M + Na) = 828.2 40-80%B; 7.040 min.; 100% 26

O CH₂ (M + Na) = 667.5 30-70%B; 8.907 min.; 96% 27

O C(O) (M + H) = 677.3 10-60%B; 10.83 min; 80% 28

O C(O) (M + H) = 609.3 10-60%B; 9.65 min; 98% 29

O C(O) (M + H) = 589.6 10-60%B; 9.52 min; 98% 30

O C(O) (M + H) = 692.3 10-60%B; 11.52 min; 98% (slow RT); 10- 60%B;10.73 min; 90% (fast RT) 31

O C(O) (M + H) = 692.3 10-60%B; 11.21 min; 98% (slow RT); 10- 60%B;10.23 min; 98% 32

O C(O) (M + H) = 658.4 10-60%B; 10.72 min; 98% 33

O C(O) (M + H) = 707.48 10-60%B; 9.9 min; 98% 34

O C(O) (M + H) = 752.6 10-60%B; 10.37 min; 98% 35

O C(O) (M + H) = 681.5 10-60%B; 8.40 min; 98% 36

O C(O) (M + H) = 666.6 10-60%B; 8.57 min; 86% 37

O C(O) (M + H) = 666.6 10-60%B; 8.70 min; 86% 38

O C(O) (M + H) = 649.6 10-60%; 9.44 min; 98% 39

O C(O) (M + H) = 669.6 10-60%B; 10.06 min; 94% 40

O C(O) (M + H) = 669.6 10-60%B; 11.0 min; 96% (slow RT); 10-60%B; 10.12min; 98% (fast RT) 41

O C(O) (M + H) = 684.6 10-60%B; 9.81 min; 95% 42

O C(O) (M + H) = 654.6 10-60%B; 9.52 min; 98% 43

O C(O) (M + H) = 654.6 10-60%B; 9.73 min; 93% 44

O C(O) (M + H) = 668.56 10-60%B; 8.35 min; 92% 45

O C(O) (M + Na) = 716.5 10-60%B; 8.86 min; 80% 46

O C(O) (M + Na) = 716.1 10-60%B; 11.26/11.58 min (1:7); 98%; 10-60%B;11.27/11.56 min (2:1); 98% 47

O C(O) (M + Na) = 678.1 10-60%B; 8.33 min; 96% 48

O C(O) (M + Na) = 697.8 10-60%B; 9.73 min; 90% 49

O C(O) (M + Na) = 647.2 10-60%B; 8.59 min; 90% 50

O C(O) (M + Na) = 660.6 10-60%B; 8.36 min; 94% 51

O C(O) (M + H) = 693.3 10-60%B; 9.42 min/10.37 min; 85% 52

O C(O) (M + H) = 700.4 10-60%B; 10.59 min; 98% 53

O C(O) (M + H) = 716.3 10-60%B; 11.24/12.18 min; 95% 54

O C(O) (M + H) = 666.4 10-60%B; 9.97 min; 98% 55

O C(O) (M + H) = 682.3 10-60%B; 9.89 min; 98% 56

O C(O) (M + H) = 696.3 10-60%B; 10.34 min; 98% 57

NH C(O) (M + H) = 676.31 20-60%B; 9.023 min.; 100% 58

NH C(O) (M + H) = 637.5 20-80%B; 5.152 min.; 100% 59

NH C(O) (M + H) = 617.5 20-80%B; 3.216 min.; 100% 60

NH C(O) (M + H) = 638.5 20-80%B; 6.221 min.; 100% 61

NH C(O) (M + H) = 588.4 20-80%B; 4.503 min.; 100% 62

NH C(O) (M + H) = 608.5 20-80%B; 5.055 min.; 100% 63

NH C(O) (M + H) = 636.5 20-80%B; 5.697 min.; 100% 64

NH C(O) C(O) (M + H) = 636.5 20-80%B; 5.548 min.; 100% 65

NH S(O)₂ (M + H) = 658.4 20-80%B; 5.632 min.; 100% 66

NH C(O) (M + H) = 658.5 20-80%B; 6.690 min.; 100% 67

NH CH₂ (M + H) = 594.5 20-80%B; 5.114 min.; 100% 68

NH C(O) (M + H) = 614.5 20-80%B; 5.559 min.; 100% 69

O CH₂ (M + H) = 560.4 20-80%B; 8.062 min.; 100% 70

O CH₂ (M + H) = 628.3 20-80%B; 9.990 min.; 100%

TABLE 2 Structures and analytical data - compounds 71-79

Z W MS Data HPLC 71

(M + H) = 869.3 40-80%B; 8.812:8.920 min.; 2:1 mix at Abu; 100% 72

(M + H) = 849.4 40-80%B; 8.380:8.539 min.; 2:1 mix at Abu; 100% 73

(M + H) = 799.5 20-60%B; 12.519 min.; 95% 74

(M + H) = 716 15.62 min.; >95% 75

(M + H) = 713 13.47 min. 76

(M + H) = 717 13.05 min.; >90% 77

(M + H) = 703 10-90%B; 8.5 min.; 8.6 min (2:1); >95% 78

(M + H) = 727 8.7 min.; 10 min. (2:1); 95% 79

(M + H) = 743 10-80%B; 5.4 min.; 95%

TABLE 3 Structures and analytical data - compounds 80-88

T W MS Data HPLC 80

(M + H) = 947 20-70%B; 6.15 min.; 95% 81

C(O)H (M + Na) = 553.60 5-45%B; 11.699 min.; 100% 82

C(O)H (M + H) = 547.4 5-45%B; 11.083 min.; 100% 83

C(O)H (M + Na) = 625.3 5-45%B; 12.258 min.; 100% 84

C(O)H (M + Na) = 626.5 5-45%B; 11.083 min.; 100% 85

C(O)H (M + Na) = 569.5 5-45%B; 11.606 min.; 100% 86

C(O)H (M + Na) = 717.2 5-45%B; 7.942 min.; 100% 87

C(O)H (M + H) = 655.3 15-55%B; 10.735 min.; 100% 88

C(O)H (M + Na) = 644.1 20-60%B; 11.360 min.; 98%

TABLE 4 Structures and analytical data - compounds 89-126

T W MS Data HPLC 89

C(O)H (M + H) = 555.9 5-45%B; 10.771 min.; 99% 90

C(O)H (M + H) = 556.0 5-45%B; 13.055 min.; 95% 91

C(O)H (M + H) = 522.4 5-45%B; 9.485 min.; 97% 92

C(O)H (M + H) = 522.55 5-45%B; 9.072 min.; 100% 93

C(O)H (M + H) = 506.33 5-45%B; 11.775 min.; 97% 94

C(O)H (M + Na) = 526.6 5-45%B; 8.822 min.; 100% 95

C(O)H (M + H) = 518. 5-45%B; 8.484 min.; 100% 96

C(O)H (M + H) = 619.6 5-45%B; 9.944 min.; 90% 97

C(O)H (M + H) = 538.7 5-45%B; 9.099 min.; 100% 98

C(O)H (M + H) = 588.6 5-45%B; 10.388 min.; 95% 99

C(O)H (M + H) = 541.1 5-45%B; 8.326 min.; 100% 100

C(O)H (M + Na) = 587.3 35-75%B; 6.763 min.; 95% 101

(M + H) = 729 10-80%B; 3.0 min; 95% 102

(M + H) = 819; (M + Na) = 840 20-70%B; 6.9 min; 95% 103

(M + H) = 848; (M + Na) = 870 20-70%B; 6.3 min; 95% 104

(M + H) = 833 20-70%B; 7.3 min; 95% 105

(M + H) = 770; (M + Na) = 792 20-70%B; 6.0 min; 95% 106

(M + H) = 801; (M + Na) = 822 20-70%; 5.9 min; 95% 107

(M + H) = 819; (M + Na) = 841 20-70%B; 3.24 min; 95% 108

(M + H) = 812; (M + Na) = 834 20-70%B; 4.9 min; 95% 109

(M + H) = 798; (M + Na) = 820 20-70%B; 4.21 min; 95% 110

(M + H) = 550 10-40%B; 7.0 min; 95% 111

(M + Na) = 886 10-50%B; 7.5 min; 95% 112

(M + H) = 638 10-80%B; 6.5 min; 95% 113

(M + H) = 865 40-80%B; 5.7 min; 95% 114

(M + H) = 669; (M + Na) = 693 25-40%B; 11.6 min; 95% 115

(M + H) = 653 10-80%B; 6.80 min; 95% 116

(M + H) = 653 10-80%B; 6.7 min; 95% 117

(M + H) = 653 10-80%B; 6.7 min; 95% 118

(M + Na) = 611 10-80%B; 5.62 min; 95% 119

(M + H) = 624 10-80%B; 12.1 min; 95% 120

(M + H) = 667 10-80%B; 13.4 min; 95% 121

(M + H) = 667 10-80%B; 13.3 min; 95% 122

(M + H) = 605 10-80%B; 11.0 min; 95% 123

(M + H) = 621 10-80%B; 9.7 min; 95% 124

(M + H) = 761 13.65 min.; 90% 125

(M + H) = 727 ND 126

(M + H) = 856 ND

TABLE 5 Structures and analytical data - compounds 127-142

M MS Data HPLC 127

(M + H) = 644.30 15-55%B; 6.08 min; 100% 128

(M + H) = 681.3 20-60%B; 8.11 min; 100% 129

(M + H) = 750.6 30-70%B; 6.99 min; 100% 130

(M + H) = 720.2 30-70%B; 6.71 min; 100% 131

(M + Na) = 715.4 30-70%B; 5.64 min; 100% 132

(M + Na) = 715.2 30-70%B; 5.58 min; 100% 133

(M + H) = 630.9 30-70%B; 3.78 min; 100% 134

(M + H) = 634.0 15-55%B; 5.90 min; 100% 135

(M + H) = 691.60 30-70%B; 4.22 min; 100% 136

(M + H) = 651.20 40-80%B; 5.59 min; 100% 137

(M + H) = 659.10 40-80%B; 4.65 min; 100% 138

(M + H) = 651.70 40-80%B; 3.83 min; 100% 139

(M + H) = 582.90 40-80%B; 2.34 min; 100% 140

(M + H) = 690.70 40-80%B; 5.15 min; 100% 141

(M + Na) = 664.80 40-80%B; 3.93 min; 100% 142

(M + Na) = 708.80 40-80%B; 5.398 min; 100%

TABLE 6 Structures and analytical data - compounds 143-197

T U MS Data HPLC 143

S(O₂) (M + Na) = 566.71 20-80%B; 10.186 min.; >95% 144

S(O₂) (M + Na) = 552.26 20-80%B; 9.985 min.; 90% 145

C(O) (M + Na) = 531.60 20-80%B; 9.978 min; 95% 146

C(O) (M + Na) = 542.37 20-80%B; 10.404 min; 95% 147

C(O) (M + Na) = 544.42 20-80%B; 10.246 min; 95% 148

C(O) (M + Na) = 454.26 20-80%B; 7.109 min; 95% 149

C(O) (M + Na) = 516.05 20-80%B; 9.668 min; 95% 150

C(O) (M + Na) = 649.17 20-80%B; 9.880 min; 95% 151

C(O) (M + Na) = 648.45 20-80%B; 10.030 min; 95% 152

C(O) (M + Na) = 587.08 20-80%B; 7.892 min; 95% 153

C(O) (M + Na) = 505.47 20-80%B; 8.583 min; 95% 154

C(O) (M + Na) = 554.96 20-80%B; 10.411 min; 95% 155

C(O) (M + Na) = 551.90 20-80%B; 6.737 min; 95% 156

C(O) (M + Na) = 566.11 20-80%B; 9.227 min; 95% 157

C(O) (M + Na) = 594.59 20-80% B; 7.567 min; 95% 158

C(O) (M + Na) = 567.00 20-80%B; 10.409 min; 95% 159

C(O) (M + Na) = 566.10 20-80%B; 10.716 min; 95% 160

C(O) (M + Na) = 559.27 20-80%B; 10.597 min; 95% 161

C(O) (M + Na) = 574.66 20-80%B; 9.723 min; 95% 162

C(O) (M + Na) = 607.43 20-80%B; 12.019 min; 95% 163

C(O) (M + H) = 514.83 20-80%B; 6.170 min; 95% 164

C(O) (M + H) = 538.87 20-80%B; 7.094 min; 99%; 20-80%B; 6.712 min; 99%165

C(O) (M + Na) = 620.77 20-80%B; 8.390 min; 99% 166

C(O) (M + H) = 536.44 20-80%B; 7.787 min; 99% 167

C(O) (M + H) = 525.58 20-80%B; 7.023 min; 99% 168

C(O) (M + Na) = 582.25 20-80%B; 7.220 min; 98% 169

C(O) (M + H) = 552.32 20-80%B; 6.410 min; 99% 170

C(O) (M + H) = 550.77 20-80%B; 6.663 min; 99% 171

C(O) (M + H) = 538.87 20-80%B; 7.101 min; 99% 172

C(O) (M + Na) = 554.79 20-80%B; 7.011 min; 99% 173

C(O) (M + H) = 551.59 20-80%B; 8.029 min; 96% 174

C(O) (M + H) = 549.86 20-80%B; 7.320 min; 99% 175

C(O) (M + Na) = 554.79 20-80%B; 6.413 min; 99% 176

C(O) (M + H) = 555.05 20-80%B; 7.065 min; 99% 177

C(O) (M + Na) = 584.55 20-80%B; 9.099 min; 99% 178

C(O) (M + H) = 535.23 20-80%B; 8.038 min; 99% 179

C(O) (M + Na) = 569.07 10-80%B; 5.885; 98% 180

C(O) (M + H) = 548.03 10-80% B; 5.991; 99% 181

C(O) (M + Na) = 533.91 10-80%B; 7.237; 99% 182

C(O) (M + Na) = 630.91 10-80%B; 9.382; 95% 183

C(O) (M + H) = 599.4 10-80% B; 7.0 min; 99% 184

C(O) (M + Na) = 545.27 10-80%B; 6.89 min; 99% 185

C(O) (M + Na) = 643.91 10-80%B; 10.43 min; 99% 186

C(O) (M + Na) = 664.69 10-80%B; 9.95 min; 99% 187

C(O) (M + Na) = 595.53 10-80%B; 8.61 min; 99% 188

C(O) (M + Na) = 596.45 10-80%B; 9.0 min; 92% 189

C(O) (M + Na) = 533.73 10-80%B; 8.438; 99% 190

C(O) (M + Na) = 554.20 10-80%B; 7.990; 99% 191

C(O) (M + Na) = 557.74 10-80%B; 9.06 min; 99% 192

C(O) (M + Na) = 545.70 10-80%B; 10.11 min; 99% 193

C(O) (M + Na) = 544.06 10-80%B; 8.41 min; 99% 194

C(O) (M + Na) = 545.49 10-80%B; 8.41 min; 96% 195

C(O) (M + Na) = 594.05 10-80%B; 8.3 min; 99% 196

C(O) (M + H) = 574.3 10-80%B; 8.84 min; 98% 197

C(O) (M + H) = 588.4 10-80%B; 9.37 min; 99%

TABLE 7 Structure and analytical data - compound 198.

MS Data HPLC 198 (M + Na) = 702.4 10-60% B; 4.2 min.; >95%

EXAMPLE 11

Insofar as compounds of formula (I) or (II) are able to inhibit NS3serine protease, they are of evident clinical utility for the treatmentof viral diseases, including HCV. These tests are predictive of thecompound's ability to inhibit HCV in vivo.

Peptides and Assays.

Peptides EDVV abuCSMSY (Abu designates-aminobutyric acid),DEMEECSQHLPYI, ECTTPCSGSWLRD and EDVV AbuC-p-nitroanilide was purchasedfrom AnaSpec Inc. (San Jose, Calif.).

Peptide content of purified, lyophilized peptides and in-house peptideswas determined by quantitative nitrogen analysis and the appropriatevalues were used in preparing stock peptide solutions

(Galbreath). pKa determinations were determined by Robertson MicrolitLaboratories, Inc. (Madison, N.J.).

HPLC cleavage assays were performed using 25 nM to 3.0 μM enzyme in 100μL volumes at 30 C containing 50 mM HEPES-KOH (pH 7.8), 100 mM NaCl, 20%glycerol, 5 mM DTT and the appropriate amount of substrate (in DMSO),with or without NS4A peptide, such that the final concentration of DMSOdid not exceed 4%. Separate control experiments verified that thispercentage of DMSO did not effect enzymatic activity. Cleavage reactionswere quenched by the addition of an equal volume of a mixture of 10%TFA:acetonitrile(1:1) and activity was assessed on a reversed phase HPLCcolumn (Rainin C18 Microsorb-MV, 5 mm, 4.6×250 mm; 0-50% acetonitrile,0.1% TFA @ 3.33% min) using a Hewlett Packard 1050 instrument withauto-injection and diode array detection at 210 nm and 280 nm (whereappropriate). Peptide elution fragments were collected and identified bymass spectrometry and N-terminal sequence analysis. Fragment identityand concentration was further verified by authentic, synthesizedproducts. Initial rates of cleavage were determined at <20% substrateconversion and catalytic parameters were determined assumingMichaelis-Menten kinetics using the MultiFit program (Day Computing,Cambridge, Mass.).

Spectrophotometric assays were run in a 96-well microtiter plate at 30C, using a SpectraMax 250 reader (Molecular Devices, Sunnyvale, Calif.)with kinetic capability. Cleavage of EDVV AbuC-p-nitroanilide (5A-pNA)substrate was performed with or without NS44 in the same buffer used forHPLC assays at 30 C, and pNA release was monitored at 405 nm. Theextinction coefficient of p-nitroaniline is independent of pH at valuesof 5.5. and above [Tuppy, H., et al., Hoppe-Seyler's Z. Physiol. Chem.,329, pp. 278-288 (1962)]; Raybuck and Luong, unpublished observations).The percentage of DMSO did not exceed 4% in these assays.

Determination of the pH dependence of Vmax, K_(m) and V_(max)/K_(m) wasperformed using a series of constant ionic strength buffers containing50 mM MES, 25 Nm Tris, 25 mM ethanolamine and 0.1 M NaCl [Morrison, J.F. and Stone, R. F., Biochemistry, 27, pp. 5499-5506 (1988)]. Theinflection point for log V data was calculated by nonlinear leastsquares fit of the data to the equation:

log v=log [Vmax/(1+H/K _(a))]

[Dixon, M. and Webb, E. C. Enzymes; Academic Press: New York; Vol., pp.138-164 (1979)]. The inflection points for log(V/K) data were calculatedby nonlinear least squares fit of the data to the equation log v=log[Vmax/(1+H/K_(a)+K_(b)/H)] [Dixon, M. and Webb, E. C. Enzymes; AcademicPress: New York; Vol., pp. 138-164 (1979)]. The program KineTic (BioKinLtd) was used in both cases.

Kinetic constants for the rapid equilibrium ordered bisubstrate reactionwere determined from rate vs [4A], [EDVV AbuC-pNA] data by non-linearleast squares fitting to equation 1 [Morrison, J. F. Biochim. Biophys.Acta, 185, pp. 269-286 (1969)] as described in the text. K_(ii) andK_(i), values for peptidyl inhibitors were determined from rate vs[inhibitor], [substrate] data and fitting to the equation for mixedinhibition:

rate=Vmax[S]/{Km(1+[I]/Kis)+[S](1+[I]/Kii)}

The commercial program KinetAsyst (StateCollege, Pa.) was used for bothprocedures. Ki values were calculated from rate vs [inhibitor] plots bya nonlinear least squares fit of the data to the equation of Morrisonfor tight binding competitive inhibition [Morrison, J. F. Biochim.Biophys. Acta, 185, pp. 269-286 (1969)]. The KineTic program (BioKinLtd) was used for this procedure.

The results are shown in Table 8. K_(i) values are expressed in μM.Category “A” indicates <1 μM inhibition; category “B” indicates 1-100 μMinhibition; category “C” indicates >100 μM. The designation “ND”indicates that the compound was not tested.

TABLE 8 Enzyme inhibition data for compounds 1-198. Cmpd. No. Ki (μM) 1B 2 B 3 B 4 B 5 B 6 B 7 B 8 B 9 B 10 B 11 B 12 B 13 B 14 B 15 B 16 B 17B 18 B 19 B 20 B 21 B 22 B 23 B 24 B 25 B 26 B 27 B 28 B 29 B 30 B 31 B32 B 33 C 34 B 35 B 36 C 37 B 38 B 39 B 40 B 41 B 42 B 43 B 44 B 45 B 46B 47 B 48 B 49 B 50 B 51 B 52 B 53 B 54 B 55 B 56 C 57 B 58 B 59 B 60 C61 C 62 B 63 B 64 B 65 B 66 B 67 C 68 C 69 B 70 B 71 A 72 B 73 B 74 B 75B 76 C 77 C 78 B 79 B 80 A 81 B 82 B 83 B 84 B 85 B 86 B 87 B 88 B 89 B90 B 91 B 92 B 93 B 94 B 95 B 96 B 97 B 98 B 99 B 100 B 101 A 102 A 103A 104 A 105 A 106 A 107 A 108 A 109 B 110 B 111 C 112 B 113 B 114 C 115B 116 B 117 B 118 B 119 C 120 B 121 C 122 C 123 B 124 B 125 B 126 C 127C 128 B 129 B 130 C 131 B 132 B 133 B 134 C 135 B 136 B 137 B 138 B 139C 140 B 141 B 142 B 143 C 144 C 145 B 146 B 147 C 148 C 149 B 150 B 151C 152 C 153 B 154 B 155 B 156 B 157 B 158 B 159 B 160 B 161 C 162 B 163C 164 C 165 C 166 C 167 C 168 B 169 C 170 C 171 C 172 C 173 C 174 B 175B 176 C 177 C 178 C 179 B 180 C 181 C 182 C 183 B 184 B 185 B 186 C 187C 188 C 189 C 190 C 191 C 192 C 193 C 194 C 195 B 196 B 197 B 198 A

While we have hereinbefore presented a number of embodiments of thisinvention, it is apparent that the basic construction can be altered toprovide other embodiments which utilize the methods of this invention.Therefore, it will be appreciated that the scope of this invention is tobe defined by the claims appended hereto rather than the specificembodiments which have been presented hereinbefore by way of example.

1-37. (canceled)
 38. A compound of the formula (II):

wherein

L is ethyl;

A¹ is R⁴ is —CH₂—CH(CH₃)₂; R⁵ and R⁶ are H; X is O or NH; Y is CH₂;C(O); C(O)(O); or S(O)₂;

A² is —CH(CH₃)₂;

M is —CH(CH₃)₂; V is NH; K is C(O) or S(O)₂; and


39. The compound according to claim 38, wherein the compound is selectedfrom any one of the following compounds:

Z X Y  1

O CH₂  2

O CH₂  3

O CH₂  4

O CH₂  5

O CH₂  6

O CH₂  7

O CH₂  8

O CH₂  9

O CH₂ 10

O CH₂ 11

O CH₂ 12

O CH₂ 13

O CH₂ 14

O CH₂ 15

O CH₂ 16

O CH₂ 17

O CH₂ 18

O CH₂ 19

O CH₂ 20

O CH₂ 21

O CH₂ 22

O CH₂ 23

O CH₂ 24

O CH₂ 25

O CH₂ 26

O CH₂ 27

O C(O) 28

O C(O) 29

O C(O) 30

O C(O) 31

O C(O) 32

O C(O) 33

O C(O) 34

O C(O) 35

O C(O) 36

O C(O) 37

O C(O) 38

O C(O) 39

O C(O) 40

O C(O) 41

O C(O) 42

O C(O) 43

O C(O) 44

O C(O) 45

O C(O) 46

O C(O) 47

O C(O) 48

O C(O) 49

O C(O) 50

O C(O) 51

O C(O) 52

O C(O) 53

O C(O) 54

O C(O) 55

O C(O) 56

O C(O) 57

NH C(O) 58

NH C(O) 59

NH C(O) 60

NH C(O) 61

NH C(O) 62

NH C(O) 63

NH C(O) 64

NH C(O) C(O) 65

NH S(O)₂ 66

NH C(O) 67

NH CH₂ 68

NH C(O) 69

O CH₂ 70

O CH₂


40. The compound according to claim 38, wherein the compound is selectedfrom any one of the following compounds:

Z W 71

72

73

74

75

76

77

78

79


41. The compound according to claim 38, wherein the compound is selectedfrom any one of the following compounds:

T W 80

81

C(O)H 82

C(O)H 83

C(O)H 84

C(O)H 85

C(O)H 86

C(O)H 87

C(O)H 88

C(O)H


42. The compound according to claim 38, wherein the compound is selectedfrom any one of the following compounds:

T W  89

C(O)H  90

C(O)H  91

C(O)H  92

C(O)H  93

C(O)H  94

C(O)H  95

C(O)H  96

C(O)H  97

C(O)H  98

C(O)H  99

C(O)H 100

C(O)H 101

102

103

104

105

106

107

108

109

110

111

112

113

114

115

116

117

118

119

120

121

122

123

124

125

126


43. The compound according to claim 38, wherein the compound is selectedfrom any one of the following compounds:

M 127

128

129

130

131

132

133

134

135

136

137

138

139

140

141

142


44. A pharmaceutically acceptable composition comprising: a) a compoundaccording to any of claims 38-43 in an amount effective to inhibit HCVNS3 protease; and b) a pharmaceutically suitable carrier.
 45. A methodfor inhibiting serine protease activity in a patient comprising the stepof administering to said patient a compound according to any one ofclaims 38-43.
 46. The method according to claim 45, wherein the serineprotease is HCV NS3 protease.
 47. A method for treating or preventing ahepatitis C viral infection in a patient comprising the step ofadministering to said patient a compound according to any one of claims38-43.
 48. The method according to claim 47, wherein said compound isadministered to said patient and is formulated together with apharmaceutically suitable carrier into a pharmaceutically acceptablecomposition.